129 results on '"Maly, DJ"'
Search Results
2. Targeting ABL-IRE1 alpha Signaling Spares ER-Stressed Pancreatic beta Cells to Reverse Autoimmune Diabetes (vol 25, pg 883, 2017)
- Author
-
Morita, S, Villalta, SA, Feldman, HC, Register, AC, Rosenthal, W, Hoffmann-Petersen, IT, Mehdizadeh, M, Ghosh, R, Wang, L, Colon-Negron, K, Meza-Acevedo, R, Backes, BJ, Maly, DJ, Bluestone, JA, and Papa, FR
- Published
- 2017
3. Targeting ABL-IRE1 alpha Signaling Spares ER- Stressed Pancreatic beta Cells to Reverse Autoimmune Diabetes
- Author
-
Morita, S, Villalta, SA, Feldman, HC, Register, AC, Rosenthal, W, Hoffmann-Petersen, IT, Mehdizadeh, M, Ghosh, R, Wang, L, Colon-Negron, K, Meza-Acevedo, R, Backes, BJ, Maly, DJ, Bluestone, JA, and Papa, FR
- Published
- 2017
4. Single-cell sensor analyses reveal signaling programs enabling Ras-G12C drug resistance.
- Author
-
Zhang JZ, Ong SE, Baker D, and Maly DJ
- Abstract
Clinical resistance to rat sarcoma virus (Ras)-G12C inhibitors is a challenge. A subpopulation of cancer cells has been shown to undergo genomic and transcriptional alterations to facilitate drug resistance but the immediate adaptive effects on Ras signaling in response to these drugs at the single-cell level is not well understood. Here, we used Ras biosensors to profile the activity and signaling environment of endogenous Ras at the single-cell level. We found that a subpopulation of KRas-G12C cells treated with Ras-G12C-guanosine-diphosphate inhibitors underwent adaptive signaling and metabolic changes driven by wild-type Ras at the Golgi and mutant KRas at the mitochondria, respectively. Our Ras biosensors identified major vault protein as a mediator of Ras activation through its scaffolding of Ras signaling pathway components and metabolite channels. Overall, methods including ours that facilitate direct analysis on the single-cell level can report the adaptations that subpopulations of cells adopt in response to cancer therapies, thus providing insight into drug resistance., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
5. A Compound That Inhibits Glycolysis in Prostate Cancer Controls Growth of Advanced Prostate Cancer.
- Author
-
Uo T, Ojo KK, Sprenger CCT, Epilepsia KS, Perera BGK, Damodarasamy M, Sun S, Kim S, Hogan HH, Hulverson MA, Choi R, Whitman GR, Barrett LK, Michaels SA, Xu LH, Sun VL, Arnold SLM, Pang HJ, Nguyen MM, Vigil ABG, Kamat V, Sullivan LB, Sweet IR, Vidadala R, Maly DJ, Van Voorhis WC, and Plymate SR
- Subjects
- Male, Humans, Animals, Mice, Cell Line, Tumor, Prostatic Neoplasms drug therapy, Prostatic Neoplasms pathology, Prostatic Neoplasms metabolism, Antineoplastic Agents pharmacology, Antineoplastic Agents therapeutic use, Glycolysis drug effects, Cell Proliferation drug effects, Xenograft Model Antitumor Assays
- Abstract
Metastatic castration-resistant prostate cancer remains incurable regardless of recent therapeutic advances. Prostate cancer tumors display highly glycolytic phenotypes as the cancer progresses. Nonspecific inhibitors of glycolysis have not been utilized successfully for chemotherapy, because of their penchant to cause systemic toxicity. This study reports the preclinical activity, safety, and pharmacokinetics of a novel small-molecule preclinical candidate, BKIDC-1553, with antiglycolytic activity. We tested a large battery of prostate cancer cell lines for inhibition of cell proliferation, in vitro. Cell-cycle, metabolic, and enzymatic assays were used to demonstrate their mechanism of action. A human patient-derived xenograft model implanted in mice and a human organoid were studied for sensitivity to our BKIDC preclinical candidate. A battery of pharmacokinetic experiments, absorption, distribution, metabolism, and excretion experiments, and in vitro and in vivo toxicology experiments were carried out to assess readiness for clinical trials. We demonstrate a new class of small-molecule inhibitors where antiglycolytic activity in prostate cancer cell lines is mediated through inhibition of hexokinase 2. These compounds display selective growth inhibition across multiple prostate cancer models. We describe a lead BKIDC-1553 that demonstrates promising activity in a preclinical xenograft model of advanced prostate cancer, equivalent to that of enzalutamide. BKIDC-1553 demonstrates safety and pharmacologic properties consistent with a compound that can be taken into human studies with expectations of a good safety margin and predicted dosing for efficacy. This work supports testing BKIDC-1553 and its derivatives in clinical trials for patients with advanced prostate cancer., (©2024 American Association for Cancer Research.)
- Published
- 2024
- Full Text
- View/download PDF
6. Multiplexed, multimodal profiling of the intracellular activity, interactions, and druggability of protein variants using LABEL-seq.
- Author
-
Simon JJ, Fowler DM, and Maly DJ
- Abstract
Multiplexed assays of variant effect are powerful tools for assessing the impact of protein sequence variation, but are limited to measuring a single protein property and often rely on indirect readouts of intracellular protein function. Here, we developed LAbeling with Barcodes and Enrichment for biochemicaL analysis by sequencing (LABEL-seq), a platform for the multimodal profiling of thousands of protein variants in cultured human cells. Multimodal measurement of ~20,000 variant effects for ~1,600 BRaf variants using LABEL-seq revealed that variation at positions that are frequently mutated in cancer had minimal effects on folding and intracellular abundance but could dramatically alter activity, protein-protein interactions, and druggability. Integrative analysis of our multimodal measurements identified networks of positions with similar roles in regulating BRaf's signaling properties and enabled predictive modeling of variant effects on complex processes such as cell proliferation and small molecule-promoted degradation. LABEL-seq provides a scalable approach for the direct measurement of multiple biochemical effects of protein variants in their native cellular context, yielding insight into protein function, disease mechanisms, and druggability.
- Published
- 2024
- Full Text
- View/download PDF
7. Profiling of drug resistance in Src kinase at scale uncovers a regulatory network coupling autoinhibition and catalytic domain dynamics.
- Author
-
Chakraborty S, Ahler E, Simon JJ, Fang L, Potter ZE, Sitko KA, Stephany JJ, Guttman M, Fowler DM, and Maly DJ
- Subjects
- Catalytic Domain, Phosphorylation, Drug Resistance, src-Family Kinases genetics, Adenosine Triphosphate metabolism
- Abstract
Kinase inhibitors are effective cancer therapies, but resistance often limits clinical efficacy. Despite the cataloging of numerous resistance mutations, our understanding of kinase inhibitor resistance is still incomplete. Here, we comprehensively profiled the resistance of ∼3,500 Src tyrosine kinase mutants to four different ATP-competitive inhibitors. We found that ATP-competitive inhibitor resistance mutations are distributed throughout Src's catalytic domain. In addition to inhibitor contact residues, residues that participate in regulating Src's phosphotransferase activity were prone to the development of resistance. Unexpectedly, we found that a resistance-prone cluster of residues located on the top face of the N-terminal lobe of Src's catalytic domain contributes to autoinhibition by reducing catalytic domain dynamics, and mutations in this cluster led to resistance by lowering inhibitor affinity and promoting kinase hyperactivation. Together, our studies demonstrate how drug resistance profiling can be used to define potential resistance pathways and uncover new mechanisms of kinase regulation., Competing Interests: Declaration of interests Authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)
- Published
- 2024
- Full Text
- View/download PDF
8. Computationally designed sensors detect endogenous Ras activity and signaling effectors at subcellular resolution.
- Author
-
Zhang JZ, Nguyen WH, Greenwood N, Rose JC, Ong SE, Maly DJ, and Baker D
- Abstract
The utility of genetically encoded biosensors for sensing the activity of signaling proteins has been hampered by a lack of strategies for matching sensor sensitivity to the physiological concentration range of the target. Here we used computational protein design to generate intracellular sensors of Ras activity (LOCKR-based Sensor for Ras activity (Ras-LOCKR-S)) and proximity labelers of the Ras signaling environment (LOCKR-based, Ras activity-dependent Proximity Labeler (Ras-LOCKR-PL)). These tools allow the detection of endogenous Ras activity and labeling of the surrounding environment at subcellular resolution. Using these sensors in human cancer cell lines, we identified Ras-interacting proteins in oncogenic EML4-Alk granules and found that Src-Associated in Mitosis 68-kDa (SAM68) protein specifically enhances Ras activity in the granules. The ability to subcellularly localize endogenous Ras activity should deepen our understanding of Ras function in health and disease and may suggest potential therapeutic strategies., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
9. Single-cell signaling analysis reveals that Major Vault Protein facilitates RasG12C inhibitor resistance.
- Author
-
Zhang JZ, Ong SE, Baker D, and Maly DJ
- Abstract
Recently developed covalent inhibitors for RasG12C provide the first pharmacological tools to target mutant Ras-driven cancers. However, the rapid development of resistance to current clinical Ras G12C inhibitors is common. Presumably, a subpopulation of RasG12C-expressing cells adapt their signaling to evade these inhibitors and the mechanisms for this phenomenon are unclear due to the lack of tools that can measure signaling with single-cell resolution. Here, we utilized recently developed Ras sensors to profile the environment of active Ras and to measure the activity of endogenous Ras in order to pair structure (Ras signalosome) to function (Ras activity), respectively, at a single-cell level. With this approach, we identified a subpopulation of KRasG12C cells treated with RasG12C-GDP inhibitors underwent oncogenic signaling and metabolic changes driven by WT Ras at the golgi and mutant Ras at the mitochondria, respectively. Our Ras sensors identified Major Vault Protein (MVP) as a mediator of Ras activation at both compartments by scaffolding Ras signaling pathway components and metabolite channels. We found that recently developed RasG12C-GTP inhibitors also led to MVP-mediated WT Ras signaling at the golgi, demonstrating that this a general mechanism RasG12C inhibitor resistance. Overall, single-cell analysis of structure-function relationships enabled the discovery of a RasG12C inhibitor-resistant subpopulation driven by MVP, providing insight into the complex and heterogenous rewiring occurring during drug resistance in cancer., Competing Interests: Declaration of interests: J.Z.Z., D.J.M., and D.B. are co-inventors in a provisional patent application (application number 63/380,884 submitted by the University of Washington) covering the biosensors described in this manuscript.
- Published
- 2023
- Full Text
- View/download PDF
10. A chemically controlled Cas9 switch enables temporal modulation of diverse effectors.
- Author
-
Wei CT, Popp NA, Peleg O, Powell RL, Borenstein E, Maly DJ, and Fowler DM
- Subjects
- Kinetics, Nucleotides, Adenine, CRISPR-Cas Systems, Gene Editing
- Abstract
CRISPR-Cas9 has yielded a plethora of effectors, including targeted transcriptional activators, base editors and prime editors. Current approaches for inducibly modulating Cas9 activity lack temporal precision and require extensive screening and optimization. We describe a versatile, chemically controlled and rapidly activated single-component DNA-binding Cas9 switch, ciCas9, which we use to confer temporal control over seven Cas9 effectors, including two cytidine base editors, two adenine base editors, a dual base editor, a prime editor and a transcriptional activator. Using these temporally controlled effectors, we analyze base editing kinetics, showing that editing occurs within hours and that rapid early editing of nucleotides predicts eventual editing magnitude. We also reveal that editing at preferred nucleotides within target sites increases the frequency of bystander edits. Thus, the ciCas9 switch offers a simple, versatile approach to generating chemically controlled Cas9 effectors, informing future effector engineering and enabling precise temporal effector control for kinetic studies., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)
- Published
- 2023
- Full Text
- View/download PDF
11. Molecular determinants of Hsp90 dependence of Src kinase revealed by deep mutational scanning.
- Author
-
Nguyen V, Ahler E, Sitko KA, Stephany JJ, Maly DJ, and Fowler DM
- Subjects
- Humans, Protein Conformation, Molecular Chaperones metabolism, Mutation, Protein Binding, src-Family Kinases genetics, src-Family Kinases metabolism, HSP90 Heat-Shock Proteins chemistry
- Abstract
Hsp90 is a molecular chaperone involved in the refolding and activation of numerous protein substrates referred to as clients. While the molecular determinants of Hsp90 client specificity are poorly understood and limited to a handful of client proteins, strong clients are thought to be destabilized and conformationally extended. Here, we measured the phosphotransferase activity of 3929 variants of the tyrosine kinase Src in both the presence and absence of an Hsp90 inhibitor. We identified 84 previously unknown functionally dependent client variants. Unexpectedly, many destabilized or extended variants were not functionally dependent on Hsp90. Instead, functionally dependent client variants were clustered in the αF pocket and β1-β2 strand regions of Src, which have yet to be described in driving Hsp90 dependence. Hsp90 dependence was also strongly correlated with kinase activity. We found that a combination of activation, global extension, and general conformational flexibility, primarily induced by variants at the αF pocket and β1-β2 strands, was necessary to render Src functionally dependent on Hsp90. Moreover, the degree of activation and flexibility required to transform Src into a functionally dependent client varied with variant location, suggesting that a combination of regulatory domain disengagement and catalytic domain flexibility are required for chaperone dependence. Thus, by studying the chaperone dependence of a massive number of variants, we highlight factors driving Hsp90 client specificity and propose a model of chaperone-kinase interactions., (© 2023 The Protein Society.)
- Published
- 2023
- Full Text
- View/download PDF
12. Multiplexed kinase interactome profiling quantifies cellular network activity and plasticity.
- Author
-
Golkowski M, Lius A, Sapre T, Lau HT, Moreno T, Maly DJ, and Ong SE
- Subjects
- Humans, Signal Transduction, Protein Interaction Maps, Neoplasms
- Abstract
Dynamic changes in protein-protein interaction (PPI) networks underlie all physiological cellular functions and drive devastating human diseases. Profiling PPI networks can, therefore, provide critical insight into disease mechanisms and identify new drug targets. Kinases are regulatory nodes in many PPI networks; yet, facile methods to systematically study kinase interactome dynamics are lacking. We describe kinobead competition and correlation analysis (kiCCA), a quantitative mass spectrometry-based chemoproteomic method for rapid and highly multiplexed profiling of endogenous kinase interactomes. Using kiCCA, we identified 1,154 PPIs of 238 kinases across 18 diverse cancer lines, quantifying context-dependent kinase interactome changes linked to cancer type, plasticity, and signaling states, thereby assembling an extensive knowledgebase for cell signaling research. We discovered drug target candidates, including an endocytic adapter-associated kinase (AAK1) complex that promotes cancer cell epithelial-mesenchymal plasticity and drug resistance. Our data demonstrate the importance of kinase interactome dynamics for cellular signaling in health and disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
13. Biolayer Interferometry Assay for Cyclin-Dependent Kinase-Cyclin Association Reveals Diverse Effects of Cdk2 Inhibitors on Cyclin Binding Kinetics.
- Author
-
Tambo CS, Tripathi S, Perera BGK, Maly DJ, Bridges AJ, Kiss G, and Rubin SM
- Subjects
- Protein Serine-Threonine Kinases metabolism, Cyclin-Dependent Kinase 2 metabolism, Cyclins metabolism, Phosphorylation, Cyclin-Dependent Kinase 4 metabolism, Cyclin-Dependent Kinases metabolism, CDC2-CDC28 Kinases metabolism
- Abstract
Cyclin-dependent kinases (CDKs) are key mediators of cell proliferation and have been a subject of oncology drug discovery efforts for over two decades. Several CDK and activator cyclin family members have been implicated in regulating the cell division cycle. While it is thought that there are canonical CDK-cyclin pairing preferences, the extent of selectivity is unclear, and increasing evidence suggests that the cell-cycle CDKs can be activated by a pool of available cyclins. The molecular details of CDK-cyclin specificity are not completely understood despite their importance for understanding cancer cell cycles and for pharmacological inhibition of cancer proliferation. We report here a biolayer interferometry assay that allows for facile quantification of CDK binding interactions with their cyclin activators. We applied this assay to measure the impact of Cdk2 inhibitors on Cyclin A (CycA) association and dissociation kinetics. We found that Type I inhibitors increase the affinity between Cdk2 and CycA by virtue of a slowed cyclin dissociation rate. In contrast, Type II inhibitors and other small-molecule Cdk2 binders have distinct effects on the CycA association and dissociation processes to decrease affinity. We propose that the differential impact of small molecules on the cyclin binding kinetics arises from the plasticity of the Cdk2 active site as the kinase transitions between active, intermediate, and inactive states.
- Published
- 2023
- Full Text
- View/download PDF
14. An essential Trypanosoma brucei protein kinase: a functional analysis of regulation and the identification of inhibitors.
- Author
-
Parsons M, Parsons B, Dean M, DeRocher AE, Islam Z, Maly DJ, and Jensen BC
- Abstract
Introduction: The protein serine/threonine kinase AEK1 is essential in the pathogenic stage of Trypanosoma brucei, the causative agent of African trypanosomiasis. AEK1 is a member of the AGC protein kinase family, although it is not closely related to a specific human AGC kinase. Our previous chemical genetic studies showed that targeted inhibition of AEK1 in parasites expressing analog-sensitive AEK1 blocked parasite growth and enhanced survival of infected mice., Methods: To further validate AEK1 as a drug target, we used the chemical genetic system to determine the effect of a 24 hour loss of AEK1 activity on cell viability at the clonal level. A panel of 429 protein kinase inhibitors were screened against the wild-type protein for binding, using time-resolved fluorescence energy transfer (TR-FRET). The role of phosphorylation sites and motifs was probed by determining whether expression of proteins harboring mutations in these sequences could rescue AEK1 conditional knockout parasites. To determine the effect that mutations in the phosphosites have on the kinase activity of cellular AEK1 we compared the in vitro kinase activity of mutant and wild-type proteins immunoprecipitated from parasite lysates using the exogenous substrate MBP. Finally, the tagged AEK1 protein was localized by deconvolution microscopy., Results: After a 24 hour exposure to an AEK1 inhibitory analog in the chemical genetic system, less than five percent of the remaining live cells can clonally expand, further validating AEK1 as a drug target. In the AEK1 inhibitor screening assay, we identified 17 hit compounds. Complementation studies showed that of the two known phosphorylation sites in the activation loop; mutation of one abolished function while mutation of the other had no discernable effect. Mutation of the other two AEK1 phosphosites gave intermediate phenotypes. Mutations in either the hydrophobic motif at the C-terminus of the protein or in the region of AEK1 predicted to bind the hydrophobic motif were also required for function. All parasites with defective AEK1 showed reduced proliferation and defects in cytokinesis, although the tested mutations differed in terms of the extent of cell death. Kinase activity of immunoprecipitated AEK1 phosphosite mutants largely paralleled the effects seen in complementation studies, although the mutation of the phosphosite adjacent to the hydrophobic motif had a greater impact on activity than predicted by the complementation studies. AEK1 was localized to cytoplasmic puncta distinct from glycosomes and acidocalcisomes., Discussion: The rapid loss of viability of cells inhibited for AEK1 supports the idea that a short course of treatment that target AEK1 may be sufficient for treatment of people or animals infected with T. brucei. Key regulatory elements between AEK1 and its closest mammalian homolog appear to be largely conserved despite the vast evolutionary distance between mammals and T. brucei. The presence of AEK1 in cytoplasmic puncta raises the possibility that its localization may also play a role in functional activity.
- Published
- 2023
- Full Text
- View/download PDF
15. ATP-competitive partial antagonists of the IRE1α RNase segregate outputs of the UPR.
- Author
-
Feldman HC, Ghosh R, Auyeung VC, Mueller JL, Kim JH, Potter ZE, Vidadala VN, Perera BGK, Olivier A, Backes BJ, Zikherman J, Papa FR, and Maly DJ
- Subjects
- Adenosine Triphosphate chemistry, Endoribonucleases metabolism, Humans, Models, Molecular, Molecular Structure, Protein Kinase Inhibitors chemistry, Protein Serine-Threonine Kinases metabolism, Protein Unfolding drug effects, Adenosine Triphosphate pharmacology, Endoribonucleases antagonists & inhibitors, Protein Kinase Inhibitors pharmacology, Protein Serine-Threonine Kinases antagonists & inhibitors
- Abstract
The unfolded protein response (UPR) homeostatically matches endoplasmic reticulum (ER) protein-folding capacity to cellular secretory needs. However, under high or chronic ER stress, the UPR triggers apoptosis. This cell fate dichotomy is promoted by differential activation of the ER transmembrane kinase/endoribonuclease (RNase) IRE1α. We previously found that the RNase of IRE1α can be either fully activated or inactivated by ATP-competitive kinase inhibitors. Here we developed kinase inhibitors, partial antagonists of IRE1α RNase (PAIRs), that partially antagonize the IRE1α RNase at full occupancy. Biochemical and structural studies show that PAIRs promote partial RNase antagonism by intermediately displacing the helix αC in the IRE1α kinase domain. In insulin-producing β-cells, PAIRs permit adaptive splicing of Xbp1 mRNA while quelling destructive ER mRNA endonucleolytic decay and apoptosis. By preserving Xbp1 mRNA splicing, PAIRs allow B cells to differentiate into immunoglobulin-producing plasma cells. Thus, an intermediate RNase-inhibitory 'sweet spot', achieved by PAIR-bound IRE1α, captures a desirable conformation for drugging this master UPR sensor/effector., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)
- Published
- 2021
- Full Text
- View/download PDF
16. Pyrrolopyrimidine Bumped Kinase Inhibitors for the Treatment of Cryptosporidiosis.
- Author
-
Hulverson MA, Choi R, Vidadala RSR, Whitman GR, Vidadala VN, Ojo KK, Barrett LK, Lynch JJ, Marsh K, Kempf DJ, Maly DJ, and Van Voorhis WC
- Subjects
- Animals, Mice, Protein Kinase Inhibitors pharmacology, Pyrimidines, Pyrroles, Antiprotozoal Agents, Cryptosporidiosis drug therapy, Cryptosporidium
- Abstract
Bumped kinase inhibitors (BKIs) that target Cryptosporidium parvum calcium-dependent protein kinase 1 have been well established as potential drug candidates against cryptosporidiosis. Recently, BKI-1649, with a 7 H -pyrrolo[2,3- d ]pyrimidin-4-amine, or "pyrrolopyrimidine", central scaffold, has shown improved efficacy in mouse models of Cryptosporidium at substantially reduced doses compared to previously explored analogs of the pyrazolopyrimidine scaffold. Here, two pyrrolopyrimidines with varied substituent groups, BKI-1812 and BKI-1814, were explored in several in vitro and in vivo models and show improvements in potency over the previously utilized pyrazolopyrimidine bumped kinase inhibitors while maintaining equivalent results in other key properties, such as toxicity and efficacy, with their pyrazolopyrimidine isosteric counterparts.
- Published
- 2021
- Full Text
- View/download PDF
17. One health therapeutics: Target-Based drug development for cryptosporidiosis and other apicomplexa diseases.
- Author
-
Van Voorhis WC, Hulverson MA, Choi R, Huang W, Arnold SLM, Schaefer DA, Betzer DP, Vidadala RSR, Lee S, Whitman GR, Barrett LK, Maly DJ, Riggs MW, Fan E, Kennedy TJ, Tzipori S, Doggett JS, Winzer P, Anghel N, Imhof D, Müller J, Hemphill A, Ferre I, Sanchez-Sanchez R, Ortega-Mora LM, and Ojo KK
- Subjects
- Animals, Apicomplexa, Humans, Antiparasitic Agents pharmacology, Cryptosporidiosis drug therapy, One Health, Piperidines pharmacology, Pyrimidines pharmacology, Quinolines pharmacology
- Abstract
This is a review of the development of bumped-kinase inhibitors (BKIs) for the therapy of One Health parasitic apicomplexan diseases. Many apicomplexan infections are shared between humans and livestock, such as cryptosporidiosis and toxoplasmosis, as well as livestock only diseases such as neosporosis. We have demonstrated proof-of-concept for BKI therapy in livestock models of cryptosporidiosis (newborn calves infected with Cryptosporidium parvum), toxoplasmosis (pregnant sheep infected with Toxoplasma gondii), and neosporosis (pregnant sheep infected with Neospora caninum). We discuss the potential uses of BKIs for the treatment of diseases caused by apicomplexan parasites in animals and humans, and the improvements that need to be made to further develop BKIs., (Copyright © 2020. Published by Elsevier B.V.)
- Published
- 2021
- Full Text
- View/download PDF
18. "Examining RAS pathway rewiring with a chemically inducible activator of RAS".
- Author
-
Rose JC, Dieter EM, Cunningham-Bryant D, and Maly DJ
- Subjects
- Cells, Cultured, Humans, Kinetics, MAP Kinase Signaling System drug effects, Protein Kinase Inhibitors chemistry, Protein Kinase Inhibitors pharmacology, Signal Transduction drug effects, ras Proteins metabolism
- Abstract
RAS signaling pathways govern diverse cellular processes, are dynamic, and exhibit marked plasticity. Yet, these features also present a considerable obstacle to their study. Here, we report the use of a recently described RAS rheostat, Chemically Inducible Activator of RAS (CIAR), to study two poorly understood phenomena in RAS biology. First, we show that short-term activation of wild type endogenous RAS can desensitize cells to EGF stimulation. Second, we examine the phenomena of paradoxical activation of RAS/ERK signaling by RAF inhibitors. Specifically, we characterize the effects on RAS/ERK signaling kinetics of four RAF inhibitors, which stabilize distinct ATP-binding site conformations. These results demonstrate the utility of CIAR in conducting quantitative studies of complex features of RAS biology.
- Published
- 2020
- Full Text
- View/download PDF
19. The Right Tool for the Job: A Chemical and Genetic Toolkit for Interrogating DCLK1 Function.
- Author
-
Fang L and Maly DJ
- Subjects
- Biology, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases metabolism, RNA Processing, Post-Transcriptional
- Abstract
Cell permeable, small molecule inhibitors are powerful tools for interrogating kinase function and validating drug targets. In this issue of Cell Chemical Biology, Liu and colleagues (2020) describe the development of a toolkit containing a highly selective DCLK1 inhibitor and complementary DCLK1 mutants for interrogating DCLK1-dependent cellular processes., (Copyright © 2020 Elsevier Ltd. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
20. Comparative assessment of the effects of bumped kinase inhibitors on early zebrafish embryo development and pregnancy in mice.
- Author
-
Anghel N, Winzer PA, Imhof D, Müller J, Langa X, Rieder J, Barrett LK, Vidadala RSR, Huang W, Choi R, Hulverson MA, Whitman GR, Arnold SL, Van Voorhis WC, Ojo KK, Maly DJ, Fan E, and Hemphill A
- Subjects
- Animals, Cell Line, Coccidiosis drug therapy, Female, Hep G2 Cells, Humans, Male, Mice, Mice, Inbred BALB C, Naphthalenes pharmacokinetics, Naphthalenes pharmacology, Neospora growth & development, Piperidines pharmacokinetics, Piperidines pharmacology, Pregnancy, Pregnancy Complications chemically induced, Protein Kinases drug effects, Protein Kinases metabolism, Pyrazoles pharmacokinetics, Pyrazoles pharmacology, Pyrimidines pharmacokinetics, Pyrimidines pharmacology, Quinolines pharmacokinetics, Quinolines pharmacology, Toxoplasma growth & development, Toxoplasmosis drug therapy, Zebrafish embryology, Embryonic Development drug effects, Naphthalenes toxicity, Neospora drug effects, Piperidines toxicity, Pyrazoles toxicity, Pyrimidines toxicity, Quinolines toxicity, Toxoplasma drug effects
- Abstract
Bumped kinase inhibitors (BKIs) are effective against a variety of apicomplexan parasites. Fifteen BKIs with promising in vitro efficacy against Neospora caninum tachyzoites, low cytotoxicity in mammalian cells, and no toxic effects in non-pregnant BALB/c mice were assessed in pregnant mice. Drugs were emulsified in corn oil and were applied by gavage for 5 days. Five BKIs did not affect pregnancy, five BKIs exhibited ~15-35% neonatal mortality and five compounds caused strong effects (infertility, abortion, stillbirth and pup mortality). Additionally, the impact of these compounds on zebrafish (Danio rerio) embryo development was assessed by exposing freshly fertilised eggs to 0.2-50 μM of BKIs and microscopic monitoring of embryo development in a blinded manner for 4 days. We propose an algorithm that includes quantification of malformations and embryo deaths, and established a scoring system that allows the calculation of an impact score (S
i ) indicating at which concentrations BKIs visibly affect zebrafish embryo development. Comparison of the two models showed that for nine compounds no clear correlation between Si and pregnancy outcome was observed. However, the three BKIs affecting zebrafish embryos only at high concentrations (≥40 μM) did not impair mouse pregnancy at all, and the three compounds that inhibited zebrafish embryo development already at 0.2 μM showed detrimental effects in the pregnancy model. Thus, the zebrafish embryo development test has limited predictive value to foresee pregnancy outcome in BKI-treated mice. We conclude that maternal health-related factors such as cardiovascular, pharmacokinetic and/or bioavailability properties also contribute to BKI-pregnancy effects., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)- Published
- 2020
- Full Text
- View/download PDF
21. Pharmacoproteomics Identifies Kinase Pathways that Drive the Epithelial-Mesenchymal Transition and Drug Resistance in Hepatocellular Carcinoma.
- Author
-
Golkowski M, Lau HT, Chan M, Kenerson H, Vidadala VN, Shoemaker A, Maly DJ, Yeung RS, Gujral TS, and Ong SE
- Subjects
- Antineoplastic Agents pharmacology, Carcinoma, Hepatocellular pathology, Humans, Liver Neoplasms pathology, Proteomics, Antineoplastic Agents therapeutic use, Carcinoma, Hepatocellular drug therapy, Drug Resistance, Neoplasm drug effects, Epithelial-Mesenchymal Transition genetics, Liver Neoplasms drug therapy, Protein Kinases metabolism
- Abstract
Hepatocellular carcinoma (HCC) is a complex and deadly disease lacking druggable genetic mutations. The limited efficacy of systemic treatments for advanced HCC implies that predictive biomarkers and drug targets are urgently needed. Most HCC drugs target protein kinases, indicating that kinase-dependent signaling networks drive HCC progression. To identify HCC signaling networks that determine responses to kinase inhibitors (KIs), we apply a pharmacoproteomics approach integrating kinome activity in 17 HCC cell lines with their responses to 299 KIs, resulting in a comprehensive dataset of pathway-based drug response signatures. By profiling patient HCC samples, we identify signatures of clinical HCC drug responses in individual tumors. Our analyses reveal kinase networks promoting the epithelial-mesenchymal transition (EMT) and drug resistance, including a FZD2-AXL-NUAK1/2 signaling module, whose inhibition reverses the EMT and sensitizes HCC cells to drugs. Our approach identifies cancer drug targets and molecular signatures of drug response for personalized oncology., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
22. Parallel Chemoselective Profiling for Mapping Protein Structure.
- Author
-
Potter ZE, Lau HT, Chakraborty S, Fang L, Guttman M, Ong SE, Fowler DM, and Maly DJ
- Subjects
- Adenosine Triphosphate chemistry, Adenosine Triphosphate metabolism, Binding, Competitive, Cysteine chemistry, HEK293 Cells, Humans, Molecular Dynamics Simulation, Mutagenesis, Site-Directed, PTEN Phosphohydrolase chemistry, PTEN Phosphohydrolase genetics, PTEN Phosphohydrolase metabolism, Protein Binding, Protein Kinase Inhibitors chemistry, Protein Kinase Inhibitors metabolism, Tandem Mass Spectrometry, Ubiquitin chemistry, Ubiquitin genetics, Ubiquitin metabolism, src-Family Kinases genetics, src-Family Kinases metabolism, Peptide Mapping methods, src-Family Kinases antagonists & inhibitors
- Abstract
Solution-based structural techniques complement high-resolution structural data by providing insight into the oft-missed links between protein structure and dynamics. Here, we present Parallel Chemoselective Profiling, a solution-based structural method for characterizing protein structure and dynamics. Our method utilizes deep mutational scanning saturation mutagenesis data to install amino acid residues with specific chemistries at defined positions on the solvent-exposed surface of a protein. Differences in the extent of labeling of installed mutant residues are quantified using targeted mass spectrometry, reporting on each residue's local environment and structural dynamics. Using our method, we studied how conformation-selective, ATP-competitive inhibitors affect the local and global structure and dynamics of full-length Src kinase. Our results highlight how parallel chemoselective profiling can be used to study a dynamic multi-domain protein, and suggest that our method will be a useful addition to the relatively small toolkit of existing protein footprinting techniques., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
23. How ATP-Competitive Inhibitors Allosterically Modulate Tyrosine Kinases That Contain a Src-like Regulatory Architecture.
- Author
-
Fang L, Vilas-Boas J, Chakraborty S, Potter ZE, Register AC, Seeliger MA, and Maly DJ
- Subjects
- Catalytic Domain, HeLa Cells, Humans, Protein Conformation, Pyrazoles metabolism, Pyrimidines metabolism, src Homology Domains, Allosteric Regulation drug effects, Protein Binding drug effects, Protein Kinase Inhibitors metabolism, src-Family Kinases antagonists & inhibitors, src-Family Kinases metabolism
- Abstract
Small molecule kinase inhibitors that stabilize distinct ATP binding site conformations can differentially modulate the global conformation of Src-family kinases (SFKs). However, it is unclear which specific ATP binding site contacts are responsible for modulating the global conformation of SFKs and whether these inhibitor-mediated allosteric effects generalize to other tyrosine kinases. Here, we describe the development of chemical probes that allow us to deconvolute which features in the ATP binding site are responsible for the allosteric modulation of the global conformation of Src. We find that the ability of an inhibitor to modulate the global conformation of Src's regulatory domain-catalytic domain module relies mainly on the influence it has on the conformation of a structural element called helix αC. Furthermore, by developing a set of orthogonal probes that target a drug-sensitized Src variant, we show that stabilizing Src's helix αC in an active conformation is sufficient to promote a Src-mediated, phosphotransferase-independent alteration in cell morphology. Finally, we report that ATP-competitive, conformation-selective inhibitors can influence the global conformation of tyrosine kinases beyond the SFKs, suggesting that the allosteric networks we observe in Src are conserved in kinases that have a similar regulatory architecture. Our study highlights that an ATP-competitive inhibitor's interactions with helix αC can have a major influence on the global conformation of some tyrosine kinases.
- Published
- 2020
- Full Text
- View/download PDF
24. Suppression of unwanted CRISPR-Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs.
- Author
-
Rose JC, Popp NA, Richardson CD, Stephany JJ, Mathieu J, Wei CT, Corn JE, Maly DJ, and Fowler DM
- Subjects
- Animals, Base Sequence, Binding Sites genetics, Biocatalysis, Cell Line, Tumor, Cells, Cultured, DNA Repair, HEK293 Cells, Humans, Mice, Models, Genetic, RNA, Guide, CRISPR-Cas Systems metabolism, CRISPR-Cas Systems, Gene Editing methods, Mutation, RNA, Guide, CRISPR-Cas Systems genetics
- Abstract
CRISPR-Cas9 nucleases are powerful genome engineering tools, but unwanted cleavage at off-target and previously edited sites remains a major concern. Numerous strategies to reduce unwanted cleavage have been devised, but all are imperfect. Here, we report that off-target sites can be shielded from the active Cas9•single guide RNA (sgRNA) complex through the co-administration of dead-RNAs (dRNAs), truncated guide RNAs that direct Cas9 binding but not cleavage. dRNAs can effectively suppress a wide-range of off-targets with minimal optimization while preserving on-target editing, and they can be multiplexed to suppress several off-targets simultaneously. dRNAs can be combined with high-specificity Cas9 variants, which often do not eliminate all unwanted editing. Moreover, dRNAs can prevent cleavage of homology-directed repair (HDR)-corrected sites, facilitating scarless editing by eliminating the need for blocking mutations. Thus, we enable precise genome editing by establishing a flexible approach for suppressing unwanted editing of both off-targets and HDR-corrected sites.
- Published
- 2020
- Full Text
- View/download PDF
25. Bumped Kinase Inhibitors as therapy for apicomplexan parasitic diseases: lessons learned.
- Author
-
Choi R, Hulverson MA, Huang W, Vidadala RSR, Whitman GR, Barrett LK, Schaefer DA, Betzer DP, Riggs MW, Doggett JS, Hemphill A, Ortega-Mora LM, McCloskey MC, Arnold SLM, Hackman RC, Marsh KC, Lynch JJ, Freiberg GM, Leroy BE, Kempf DJ, Choy RKM, de Hostos EL, Maly DJ, Fan E, Ojo KK, and Van Voorhis WC
- Subjects
- Animals, Apicomplexa metabolism, Cryptosporidiosis drug therapy, Cryptosporidium drug effects, Cryptosporidium metabolism, Humans, Protein Kinases drug effects, Protein Kinases metabolism, Toxoplasma drug effects, Toxoplasma metabolism, Toxoplasmosis drug therapy, Apicomplexa drug effects, Coccidiosis drug therapy, Protein Kinase Inhibitors adverse effects, Protein Kinase Inhibitors chemistry, Protein Kinase Inhibitors pharmacology
- Abstract
Bumped Kinase Inhibitors, targeting Calcium-dependent Protein Kinase 1 in apicomplexan parasites with a glycine gatekeeper, are promising new therapeutics for apicomplexan diseases. Here we will review advances, as well as challenges and lessons learned regarding efficacy, safety, and pharmacology that have shaped our selection of pre-clinical candidates., (Copyright © 2020. Published by Elsevier Ltd.)
- Published
- 2020
- Full Text
- View/download PDF
26. Kinobead/LC-MS Phosphokinome Profiling Enables Rapid Analyses of Kinase-Dependent Cell Signaling Networks.
- Author
-
Golkowski M, Vidadala VN, Lau HT, Shoemaker A, Shimizu-Albergine M, Beavo J, Maly DJ, and Ong SE
- Subjects
- Chromatography, Liquid, Humans, Male, Phosphorylation, Protein Kinases metabolism, Signal Transduction, Tandem Mass Spectrometry
- Abstract
Kinase-catalyzed protein phosphorylation is fundamental to eukaryotic signal transduction, regulating most cellular processes. Kinases are frequently dysregulated in cancer, inflammation, and degenerative diseases, and because they can be inhibited with small molecules, they became important drug targets. Accordingly, analytical approaches that determine kinase activation states are critically important to understand kinase-dependent signal transduction and to identify novel drug targets and predictive biomarkers. Multiplexed inhibitor beads (MIBs or kinobeads) efficiently enrich kinases from cell lysates for liquid chromatography-mass spectrometry (LC-MS) analysis. When combined with phosphopeptide enrichment, kinobead/LC-MS can also quantify the phosphorylation state of kinases, which determines their activation state. However, an efficient kinobead/LC-MS kinase phospho-profiling protocol that allows routine analyses of cell lines and tissues has not yet been developed. Here, we present a facile workflow that quantifies the global phosphorylation state of kinases with unprecedented sensitivity. We also found that our kinobead/LC-MS protocol can measure changes in kinase complex composition and show how these changes can indicate kinase activity. We demonstrate the utility of our approach in specifying kinase signaling pathways that control the acute steroidogenic response in Leydig cells; this analysis establishes the first comprehensive framework for the post-translational control of steroid biosynthesis.
- Published
- 2020
- Full Text
- View/download PDF
27. A chemically-controlled system for activating RAS GTPases.
- Author
-
Dieter EM and Maly DJ
- Subjects
- Signal Transduction, ras Proteins genetics, ras Proteins metabolism
- Abstract
RAS GTPases are involved in a number of dynamic signaling processes and have been a major focus of research due to the prevalence of activating RAS mutations in cancer. However, despite decades of research, some fundamental aspects of RAS biology are still not well understood. Difficulty in fully defining RAS-driven signaling stems from the overall complexity of downstream pathways and a lack of tools for specifically perturbing RAS function. To better characterize RAS-driven signaling, we recently developed a chemical genetic system for activating endogenous RAS with a small molecule. In this chapter, we describe the use of chemically inducible activator of RAS (CIAR), a single-protein, chemical genetic system that allows the rapid and dose-dependent activation of endogenous RAS. Methods in this chapter also describe the validation of RAS activation with CIAR through the analysis of downstream signaling., (© 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
28. Temporal and rheostatic control of genome editing with a chemically-inducible Cas9.
- Author
-
Wei CT, Maly DJ, and Fowler DM
- Subjects
- DNA Breaks, Double-Stranded, DNA Cleavage, Endonucleases genetics, CRISPR-Cas Systems, Gene Editing
- Abstract
Nuclease-mediated DNA cleavage and subsequent repair lie at the heart of genome editing, and the RNA-guided endonuclease Cas9 has emerged as the most widely-used tool for facilitating this process. Extensive biochemical and biophysical efforts have revealed much regarding the structure, mechanism, and cellular properties of Cas9. This has enabled engineering of Cas9 variants with enhanced activity, specificity, and other features. However, we lack a detailed understanding of the kinetics of Cas9-mediated DNA cleavage and repair in vivo. To study in vivo Cas9 cleavage kinetics and activity dose-dependence, we have engineered a chemically-inducible, single-component Cas9, ciCas9. ciCas9 allows for temporal and rheostatic control of Cas9 activity using a small molecule activator, A115. We have also developed a droplet-digital PCR-based assay (DSB-ddPCR) to directly quantify Cas9-mediated double-stranded breaks (DSBs). The methods in this chapter describe the application of ciCas9 and DSB-ddPCR to study the kinetics and dose-dependence of Cas9 editing in vivo., (© 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
29. Subcellular drug targeting illuminates local kinase action.
- Author
-
Bucko PJ, Lombard CK, Rathbun L, Garcia I, Bhat A, Wordeman L, Smith FD, Maly DJ, Hehnly H, and Scott JD
- Subjects
- Animals, Aurora Kinase A chemistry, Cell Cycle Proteins chemistry, Centrosome chemistry, Centrosome ultrastructure, Embryonic Development genetics, Gene Expression Regulation, Developmental, Humans, Kinetochores chemistry, Microtubules genetics, Phosphorylation genetics, Protein Kinase Inhibitors chemistry, Protein Serine-Threonine Kinases chemistry, Proto-Oncogene Proteins chemistry, Zebrafish genetics, Zebrafish growth & development, Polo-Like Kinase 1, A Kinase Anchor Proteins genetics, Aurora Kinase A genetics, Cell Cycle Proteins genetics, Mitosis genetics, Protein Serine-Threonine Kinases genetics, Proto-Oncogene Proteins genetics
- Abstract
Deciphering how signaling enzymes operate within discrete microenvironments is fundamental to understanding biological processes. A-kinase anchoring proteins (AKAPs) restrict the range of action of protein kinases within intracellular compartments. We exploited the AKAP targeting concept to create genetically encoded platforms that restrain kinase inhibitor drugs at distinct subcellular locations. Lo cal K inase I nhibition ( LoKI ) allows us to ascribe organelle-specific functions to broad specificity kinases. Using chemical genetics, super resolution microscopy, and live-cell imaging we discover that centrosomal delivery of Polo-like kinase 1 (Plk1) and Aurora A (AurA) inhibitors attenuates kinase activity, produces spindle defects, and prolongs mitosis. Targeted inhibition of Plk1 in zebrafish embryos illustrates how centrosomal Plk1 underlies mitotic spindle assembly. Inhibition of kinetochore-associated pools of AurA blocks phosphorylation of microtubule-kinetochore components. This versatile precision pharmacology tool enhances investigation of local kinase biology., Competing Interests: PB, CL, LR, IG, AB, LW, FS, DM, HH, JS No competing interests declared, (© 2019, Bucko et al.)
- Published
- 2019
- Full Text
- View/download PDF
30. Development of a Chemical Toolset for Studying the Paralog-Specific Function of IRE1.
- Author
-
Feldman HC, Vidadala VN, Potter ZE, Papa FR, Backes BJ, and Maly DJ
- Subjects
- Allosteric Regulation, Animals, Endoplasmic Reticulum Stress, Endoribonucleases metabolism, Humans, Protein Serine-Threonine Kinases metabolism, Ribonucleases metabolism, Endoribonucleases physiology, Protein Serine-Threonine Kinases physiology
- Abstract
The dual kinase endoribonuclease IRE1 is a master regulator of cell fate decisions in cells experiencing endoplasmic reticulum (ER) stress. In mammalian cells, there are two paralogs of IRE1: IRE1α and IRE1β. While IRE1α has been extensively studied, much less is understood about IRE1β and its role in signaling. In addition, whether the regulation of IRE1β's enzymatic activities varies compared to IRE1α is not known. Here, we show that the RNase domain of IRE1β is enzymatically active and capable of cleaving an XBP1 RNA mini-substrate in vitro . Using ATP-competitive inhibitors, we find that, like IRE1α, there is an allosteric relationship between the kinase and RNase domains of IRE1β. This allowed us to develop a novel toolset of both paralog specific and dual-IRE1α/β kinase inhibitors that attenuate RNase activity (KIRAs). Using sequence alignments of IRE1α and IRE1β, we propose a model for paralog-selective inhibition through interactions with nonconserved residues that differentiate the ATP-binding pockets of IRE1α and IRE1β.
- Published
- 2019
- Full Text
- View/download PDF
31. Parallel Signaling through IRE1α and PERK Regulates Pancreatic Neuroendocrine Tumor Growth and Survival.
- Author
-
Moore PC, Qi JY, Thamsen M, Ghosh R, Peng J, Gliedt MJ, Meza-Acevedo R, Warren RE, Hiniker A, Kim GE, Maly DJ, Backes BJ, Papa FR, and Oakes SA
- Subjects
- Adenine analogs & derivatives, Adenine pharmacology, Adenine therapeutic use, Animals, Cell Line, Tumor, Cell Proliferation drug effects, Cell Survival drug effects, Disease Models, Animal, Endoplasmic Reticulum Stress drug effects, Endoribonucleases metabolism, Female, Humans, Indoles pharmacology, Indoles therapeutic use, Mice, Mice, Transgenic, Neuroendocrine Tumors genetics, Neuroendocrine Tumors pathology, Pancreatic Neoplasms genetics, Pancreatic Neoplasms pathology, Protein Kinase Inhibitors therapeutic use, Protein Serine-Threonine Kinases metabolism, Signal Transduction drug effects, Unfolded Protein Response drug effects, Xenograft Model Antitumor Assays, eIF-2 Kinase metabolism, Endoribonucleases antagonists & inhibitors, Neuroendocrine Tumors drug therapy, Pancreatic Neoplasms drug therapy, Protein Kinase Inhibitors pharmacology, Protein Serine-Threonine Kinases antagonists & inhibitors, eIF-2 Kinase antagonists & inhibitors
- Abstract
Master regulators of the unfolded protein response (UPR), IRE1α and PERK, promote adaptation or apoptosis depending on the level of endoplasmic reticulum (ER) stress. Although the UPR is activated in many cancers, its effects on tumor growth remain unclear. Derived from endocrine cells, pancreatic neuroendocrine tumors (PanNET) universally hypersecrete one or more peptide hormones, likely sensitizing these cells to high ER protein-folding stress. To assess whether targeting the UPR is a viable therapeutic strategy, we analyzed human PanNET samples and found evidence of elevated ER stress and UPR activation. Genetic and pharmacologic modulation of IRE1α and PERK in cultured cells, xenograft, and spontaneous genetic (RIP-Tag2) mouse models of PanNETs revealed that UPR signaling was optimized for adaptation and that inhibiting either IRE1α or PERK led to hyperactivation and apoptotic signaling through the reciprocal arm, thereby halting tumor growth and survival. These results provide a strong rationale for therapeutically targeting the UPR in PanNETs and other cancers with elevated ER stress. SIGNIFICANCE: The UPR is upregulated in pancreatic neuroendocrine tumors and its inhibition significantly reduces tumor growth in preclinical models, providing strong rationale for targeting the UPR in these cancers., (©2019 American Association for Cancer Research.)
- Published
- 2019
- Full Text
- View/download PDF
32. Multi-input chemical control of protein dimerization for programming graded cellular responses.
- Author
-
Foight GW, Wang Z, Wei CT, Jr Greisen P, Warner KM, Cunningham-Bryant D, Park K, Brunette TJ, Sheffler W, Baker D, and Maly DJ
- Subjects
- Animals, Cell Line, Combinatorial Chemistry Techniques, Drug Design, HeLa Cells, Humans, Mice, Models, Molecular, NIH 3T3 Cells, Optogenetics methods, Protein Conformation, Protein Multimerization, Protein Processing, Post-Translational, Signal Transduction, Synthetic Biology methods, Proteins chemistry, Proteins metabolism
- Abstract
Chemical and optogenetic methods for post-translationally controlling protein function have enabled modulation and engineering of cellular functions. However, most of these methods only confer single-input, single-output control. To increase the diversity of post-translational behaviors that can be programmed, we built a system based on a single protein receiver that can integrate multiple drug inputs, including approved therapeutics. Our system translates drug inputs into diverse outputs using a suite of engineered reader proteins to provide variable dimerization states of the receiver protein. We show that our single receiver protein architecture can be used to program a variety of cellular responses, including graded and proportional dual-output control of transcription and mammalian cell signaling. We apply our tools to titrate the competing activities of the Rac and Rho GTPases to control cell morphology. Our versatile tool set will enable researchers to post-translationally program mammalian cellular processes and to engineer cell therapies.
- Published
- 2019
- Full Text
- View/download PDF
33. P-Glycoprotein-Mediated Efflux Reduces the In Vivo Efficacy of a Therapeutic Targeting the Gastrointestinal Parasite Cryptosporidium.
- Author
-
Arnold SLM, Choi R, Hulverson MA, Whitman GR, Mccloskey MC, Dorr CS, Vidadala RSR, Khatod M, Morada M, Barrett LK, Maly DJ, Yarlett N, and Van Voorhis WC
- Subjects
- Animals, Biological Transport, Active, Caco-2 Cells, Cell Membrane Permeability drug effects, Cryptosporidiosis parasitology, Disease Models, Animal, Drug Discovery methods, Enterocytes drug effects, Enterocytes metabolism, Enterocytes parasitology, Female, Gastrointestinal Absorption drug effects, Humans, Interferon-gamma genetics, Mice, Mice, Knockout, Naphthalenes chemistry, Piperidines chemistry, Pyrazoles chemistry, Pyrimidines chemistry, Quinolines chemistry, Treatment Outcome, ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism, Cryptosporidiosis drug therapy, Cryptosporidium drug effects, Intestinal Diseases, Parasitic drug therapy, Naphthalenes metabolism, Naphthalenes therapeutic use, Piperidines metabolism, Piperidines therapeutic use, Pyrazoles metabolism, Pyrazoles therapeutic use, Pyrimidines metabolism, Pyrimidines therapeutic use, Quinolines metabolism, Quinolines therapeutic use
- Abstract
Recent studies have illustrated the burden Cryptosporidium infection places on the lives of malnourished children and immunocompromised individuals. Treatment options remain limited, and efforts to develop a new therapeutic are currently underway. However, there are unresolved questions about the ideal pharmacokinetic characteristics of new anti-Cryptosporidium therapeutics. Specifically, should drug developers optimize therapeutics and formulations to increase drug exposure in the gastrointestinal lumen, enterocytes, or systemic circulation? Furthermore, how should researchers interpret data suggesting their therapeutic is a drug efflux transporter substrate? In vivo drug transporter-mediated alterations in efficacy are well recognized in multiple disease areas, but the impact of intestinal transporters on therapeutic efficacy against enteric diseases has not been established. Using multiple in vitro models and a mouse model of Cryptosporidium infection, we characterized the effect of P-glycoprotein efflux on bumped kinase inhibitor pharmacokinetics and efficacy. Our results demonstrated P-glycoprotein decreases bumped kinase inhibitor enterocyte exposure, resulting in reduced in vivo efficacy against Cryptosporidium. Furthermore, a hollow fiber model of Cryptosporidium infection replicated the in vivo impact of P-glycoprotein on anti-Cryptosporidium efficacy. In conclusion, when optimizing drug candidates targeting the gastrointestinal epithelium or gastrointestinal epithelial infections, drug developers should consider the adverse impact of active efflux transporters on efficacy., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)
- Published
- 2019
- Full Text
- View/download PDF
34. Bumped kinase inhibitor 1369 is effective against Cystoisospora suis in vivo and in vitro.
- Author
-
Shrestha A, Ojo KK, Koston F, Ruttkowski B, Vidadala RSR, Dorr CS, Navaluna ED, Whitman GR, Barrett KF, Barrett LK, Hulverson MA, Choi R, Michaels SA, Maly DJ, Hemphill A, Van Voorhis WC, and Joachim A
- Subjects
- Animals, Antiprotozoal Agents chemistry, Coccidiosis drug therapy, Coccidiosis parasitology, Female, Male, Protein Kinase Inhibitors chemistry, Protein Kinases metabolism, Protozoan Proteins antagonists & inhibitors, Protozoan Proteins metabolism, Sarcocystidae enzymology, Sarcocystidae growth & development, Swine, Swine Diseases drug therapy, Antiprotozoal Agents administration & dosage, Coccidiosis veterinary, Protein Kinase Inhibitors administration & dosage, Sarcocystidae drug effects, Swine Diseases parasitology
- Abstract
Cystoisosporosis is a leading diarrheal disease in suckling piglets. With the confirmation of resistance against the only available drug toltrazuril, there is a substantial need for novel therapeutics to combat the infection and its negative effects on animal health. In closely related apicomplexan species, bumped kinase inhibitors (BKIs) targeting calcium-dependent protein kinase 1 (CDPK1) were shown to be effective in inhibiting host-cell invasion and parasite growth. Therefore, the gene coding for Cystoisospora suis CDPK1 (CsCDPK1) was identified and cloned to investigate activity and thermal stabilization of the recombinant CsCDPK1 enzyme by BKI 1369. In this comprehensive study, the efficacy, safety and pharmacokinetics of BKI 1369 in piglets experimentally infected with Cystoisospora suis (toltrazuril-sensitive, Wien-I and toltrazuril-resistant, Holland-I strains) were determined in vivo and in vitro using an established animal infection model and cell culture, respectively. BKI 1369 inhibited merozoite proliferation in intestinal porcine epithelial cells-1 (IPEC-1) by at least 50% at a concentration of 40 nM, and proliferation was almost completely inhibited (>95%) at 200 nM. Nonetheless, exposure of infected cultures to 200 nM BKI 1369 for five days did not induce structural alterations in surviving merozoites as confirmed by transmission electron microscopy. Five-day treatment with BKI 1369 (10 mg/kg BW twice a day) effectively suppressed oocyst excretion and diarrhea and improved body weight gains in treated piglets without obvious side effects for both toltrazuril-sensitive, Wien-I and resistant, Holland-I C. suis strains. The plasma concentration of BKI 1369 in piglets increased to 11.7 μM during treatment, suggesting constant drug accumulation and exposure of parasites to the drug. Therefore, oral applications of BKI 1369 could potentially be a therapeutic alternative against porcine cystoisosporosis. For use in pigs, future studies on BKI 1369 should be directed towards ease of drug handling and minimizing treatment frequencies., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
35. Chemoproteomic Method for Profiling Inhibitor-Bound Kinase Complexes.
- Author
-
Fang L, Chakraborty S, Dieter EM, Potter ZE, Lombard CK, and Maly DJ
- Subjects
- Cyclooctanes chemistry, HEK293 Cells, Humans, Molecular Structure, Protein Kinase Inhibitors chemistry, src-Family Kinases chemistry, src-Family Kinases metabolism, Cyclooctanes pharmacology, Protein Kinase Inhibitors pharmacology, src-Family Kinases antagonists & inhibitors
- Abstract
Small molecule inhibitors often only block a subset of the cellular functions of their protein targets. In many cases, how inhibiting only a portion of a multifunctional protein's functions affects the state of the cell is not well-understood. Therefore, tools that allow the systematic characterization of the cellular interactions that inhibitor-bound proteins make would be of great utility, especially for multifunctional proteins. Here, we describe a chemoproteomic strategy for interrogating the cellular localization and interactomes of inhibitor-bound kinases. By developing a set of orthogonal inhibitors that contain a trans -cyclooctene (TCO) click handle, we are able to enrich and characterize the proteins complexed to a drug-sensitized variant of the multidomain kinase Src. We show that Src's cellular interactions are highly influenced by the intermolecular accessibility of its regulatory domains, which can be allosterically modulated through its ATP-binding site. Furthermore, we find that the signaling status of the cell also has a large effect on Src's interactome. Finally, we demonstrate that our TCO-conjugated probes can be used as a part of a proximity ligation assay to study Src's localization and interactions in situ . Together, our chemoproteomic strategy represents a comprehensive method for studying the localization and interactomes of inhibitor-bound kinases and, potentially, other druggable protein targets.
- Published
- 2019
- Full Text
- View/download PDF
36. Targeting Dynamic ATP-Binding Site Features Allows Discrimination between Highly Homologous Protein Kinases.
- Author
-
Chakraborty S, Inukai T, Fang L, Golkowski M, and Maly DJ
- Subjects
- Binding Sites, Phosphorylation, Protein Binding, Protein Conformation, Protein Domains, Protein Kinase Inhibitors pharmacology, src-Family Kinases antagonists & inhibitors, src-Family Kinases chemistry, Adenosine Triphosphate metabolism, src-Family Kinases metabolism
- Abstract
ATP-competitive inhibitors that demonstrate exquisite selectivity for specific members of the human kinome have been developed. Despite this success, the identification of highly selective inhibitors is still very challenging, and it is often not possible to rationally engineer selectivity between the ATP-binding sites of kinases, especially among closely related family members. Src-family kinases (SFKs) are a highly homologous family of eight multidomain, nonreceptor tyrosine kinases that play general and specialized roles in numerous cellular processes. The high sequence and functional similarities between SFK members make it hard to rationalize how selectivity can be gained with inhibitors that target the ATP-binding site. Here, we describe the development of a series of inhibitors that are highly selective for the ATP-binding sites of the SFKs Lyn and Hck over other SFKs. By biochemically characterizing how these selective ATP-competitive inhibitors allosterically influence the global conformation of SFKs, we demonstrate that they most likely interact with a binding pocket created by the movement of the conformationally flexible helix αC in the ATP-binding site. With a series of sequence swap experiments, we show that sensitivity to this class of selective inhibitors is due to the identity of residues that control the conformational flexibility of helix αC rather than any specific ATP-binding site interactions. Thus, the ATP-binding sites of highly homologous kinases can be discriminated by targeting heterogeneity within conformationally flexible regions.
- Published
- 2019
- Full Text
- View/download PDF
37. A Combined Approach Reveals a Regulatory Mechanism Coupling Src's Kinase Activity, Localization, and Phosphotransferase-Independent Functions.
- Author
-
Ahler E, Register AC, Chakraborty S, Fang L, Dieter EM, Sitko KA, Vidadala RSR, Trevillian BM, Golkowski M, Gelman H, Stephany JJ, Rubin AF, Merritt EA, Fowler DM, and Maly DJ
- Subjects
- Allosteric Regulation genetics, Cell Membrane chemistry, Cell Membrane enzymology, HEK293 Cells, Humans, Phosphorylation, src-Family Kinases genetics, Catalytic Domain genetics, Mutagenesis genetics, Protein Conformation, src-Family Kinases chemistry
- Abstract
Multiple layers of regulation modulate the activity and localization of protein kinases. However, many details of kinase regulation remain incompletely understood. Here, we apply saturation mutagenesis and a chemical genetic method for allosterically modulating kinase global conformation to Src kinase, providing insight into known regulatory mechanisms and revealing a previously undiscovered interaction between Src's SH4 and catalytic domains. Abrogation of this interaction increased phosphotransferase activity, promoted membrane association, and provoked phosphotransferase-independent alterations in cell morphology. Thus, Src's SH4 domain serves as an intramolecular regulator coupling catalytic activity, global conformation, and localization, as well as mediating a phosphotransferase-independent function. Sequence conservation suggests that the SH4 domain regulatory interaction exists in other Src-family kinases. Our combined approach's ability to reveal a regulatory mechanism in one of the best-studied kinases suggests that it could be applied broadly to provide insight into kinase structure, regulation, and function., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
38. Pharmacokinetics and In Vivo Efficacy of Pyrazolopyrimidine, Pyrrolopyrimidine, and 5-Aminopyrazole-4-Carboxamide Bumped Kinase Inhibitors against Toxoplasmosis.
- Author
-
Hulverson MA, Bruzual I, McConnell EV, Huang W, Vidadala RSR, Choi R, Arnold SLM, Whitman GR, McCloskey MC, Barrett LK, Rivas KL, Scheele S, DeRocher AE, Parsons M, Ojo KK, Maly DJ, Fan E, Van Voorhis WC, and Doggett JS
- Subjects
- Administration, Oral, Animals, Area Under Curve, Female, In Vitro Techniques, Mice, Protein Kinase Inhibitors blood, Protein Kinase Inhibitors pharmacology, Pyrazoles blood, Pyrazoles pharmacology, Pyrimidines blood, Pyrimidines pharmacology, Protein Kinase Inhibitors therapeutic use, Protozoan Proteins antagonists & inhibitors, Pyrazoles therapeutic use, Pyrimidines therapeutic use, Toxoplasmosis, Animal drug therapy, Toxoplasmosis, Cerebral drug therapy
- Abstract
Bumped kinase inhibitors (BKIs) have been shown to be potent inhibitors of Toxoplasma gondii calcium-dependent protein kinase 1. Pyrazolopyrimidine and 5-aminopyrazole-4-carboxamide scaffold-based BKIs are effective in acute and chronic experimental models of toxoplasmosis. Through further exploration of these 2 scaffolds and a new pyrrolopyrimidine scaffold, additional compounds have been identified that are extremely effective against acute experimental toxoplasmosis. The in vivo efficacy of these BKIs demonstrates that the cyclopropyloxynaphthyl, cyclopropyloxyquinoline, and 2-ethoxyquinolin-6-yl substituents are associated with efficacy across scaffolds. In addition, a broad range of plasma concentrations after oral dosing resulted from small structural changes to the BKIs. These select BKIs include anti-Toxoplasma compounds that are effective against acute experimental toxoplasmosis and are not toxic in human cell assays, nor to mice when administered for therapy. The BKIs described here are promising late leads for improving anti-Toxoplasma therapy., (Published by Oxford University Press for the Infectious Diseases Society of America 2018.)
- Published
- 2019
- Full Text
- View/download PDF
39. A Chemically Disrupted Proximity System for Controlling Dynamic Cellular Processes.
- Author
-
Cunningham-Bryant D, Dieter EM, Foight GW, Rose JC, Loutey DE, and Maly DJ
- Subjects
- Cell Physiological Phenomena, HEK293 Cells, Hepacivirus drug effects, Hepacivirus enzymology, Humans, Protease Inhibitors chemistry, Viral Nonstructural Proteins chemistry, Viral Nonstructural Proteins metabolism, Protease Inhibitors pharmacology, Viral Nonstructural Proteins antagonists & inhibitors
- Abstract
Chemical methods that allow the spatial proximity of proteins to be temporally modulated are powerful tools for studying biology and engineering synthetic cellular behaviors. Here, we describe a new chemically controlled method for rapidly disrupting the interaction between two basally colocalized protein binding partners. Our chemically disrupted proximity (CDP) system is based on the interaction between the hepatitis C virus protease (HCVp) NS3a and a genetically encoded peptide inhibitor. Using clinically approved antiviral inhibitors as chemical disrupters of the NS3a/peptide interaction, we demonstrate that our CDP system can be used to confer temporal control over diverse intracellular processes. This NS3a-based CDP system represents a new modality for engineering chemical control over intracellular protein function that is complementary to currently available techniques.
- Published
- 2019
- Full Text
- View/download PDF
40. Small molecule inhibition of IRE1α kinase/RNase has anti-fibrotic effects in the lung.
- Author
-
Thamsen M, Ghosh R, Auyeung VC, Brumwell A, Chapman HA, Backes BJ, Perara G, Maly DJ, Sheppard D, and Papa FR
- Subjects
- Alveolar Epithelial Cells metabolism, Alveolar Epithelial Cells pathology, Animals, Apoptosis drug effects, Cell Line, Endoplasmic Reticulum Stress drug effects, Fibrosis metabolism, Fibrosis pathology, Lung metabolism, Lung pathology, Mice, Protein Kinase Inhibitors therapeutic use, Unfolded Protein Response drug effects, Alveolar Epithelial Cells drug effects, Endoribonucleases antagonists & inhibitors, Fibrosis drug therapy, Lung drug effects, Protein Kinase Inhibitors pharmacology, Protein Serine-Threonine Kinases antagonists & inhibitors
- Abstract
Endoplasmic reticulum stress (ER stress) has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a disease of progressive fibrosis and respiratory failure. ER stress activates a signaling pathway called the unfolded protein response (UPR) that either restores homeostasis or promotes apoptosis. The bifunctional kinase/RNase IRE1α is a UPR sensor/effector that promotes apoptosis if ER stress remains high and irremediable (i.e., a "terminal" UPR). Using multiple small molecule inhibitors against IRE1α, we show that ER stress-induced apoptosis of murine alveolar epithelial cells can be mitigated in vitro. In vivo, we show that bleomycin exposure to murine lungs causes early ER stress to activate IRE1α and the terminal UPR prior to development of pulmonary fibrosis. Small-molecule IRE1α kinase-inhibiting RNase attenuators (KIRAs) that we developed were used to evaluate the contribution of IRE1α activation to bleomycin-induced pulmonary fibrosis. One such KIRA-KIRA7-provided systemically to mice at the time of bleomycin exposure decreases terminal UPR signaling and prevents lung fibrosis. Administration of KIRA7 14 days after bleomycin exposure even promoted the reversal of established fibrosis. Finally, we show that KIRA8, a nanomolar-potent, monoselective KIRA compound derived from a completely different scaffold than KIRA7, likewise promoted reversal of established fibrosis. These results demonstrate that IRE1α may be a promising target in pulmonary fibrosis and that kinase inhibitors of IRE1α may eventually be developed into efficacious anti-fibrotic drugs., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2019
- Full Text
- View/download PDF
41. Allosteric Modulation of JNK Docking Site Interactions with ATP-Competitive Inhibitors.
- Author
-
Lombard CK, Davis AL, Inukai T, and Maly DJ
- Subjects
- Animals, Binding Sites, Catalytic Domain, Humans, Protein Structure, Secondary, Adenosine Triphosphate analogs & derivatives, Adenosine Triphosphate chemistry, JNK Mitogen-Activated Protein Kinases antagonists & inhibitors, JNK Mitogen-Activated Protein Kinases chemistry, MAP Kinase Signaling System, Molecular Docking Simulation, Protein Kinase Inhibitors chemistry
- Abstract
The c-Jun N-terminal kinases (JNKs) play a wide variety of roles in cellular signaling processes, dictating important, and even divergent, cellular fates. These essential kinases possess docking surfaces distal to their active sites that interact with diverse binding partners, including upstream activators, downstream substrates, and protein scaffolds. Prior studies have suggested that the interactions of certain protein-binding partners with one such JNK docking surface, termed the D-recruitment site (DRS), can allosterically influence the conformational state of the ATP-binding pocket of JNKs. To further explore the allosteric relationship between the ATP-binding pockets and DRSs of JNKs, we investigated how the interactions of the scaffolding protein JIP1, as well as the upstream activators MKK4 and MKK7, are allosterically influenced by the ATP-binding site occupancy of the JNKs. We show that the affinity of the JNKs for JIP1 can be divergently modulated with ATP-competitive inhibitors, with a >50-fold difference in dissociation constant observed between the lowest- and highest-affinity JNK1-inhibitor complexes. Furthermore, we found that we could promote or attenuate phosphorylation of JNK1's activation loop by MKK4 and MKK7, by varying the ATP-binding site occupancy. Given that JIP1, MKK4, and MKK7 all interact with JNK DRSs, these results demonstrate that there is functional allostery between the ATP-binding sites and DRSs of these kinases. Furthermore, our studies suggest that ATP-competitive inhibitors can allosterically influence the intracellular binding partners of the JNKs.
- Published
- 2018
- Full Text
- View/download PDF
42. Toxoplasma Calcium-Dependent Protein Kinase 1 Inhibitors: Probing Activity and Resistance Using Cellular Thermal Shift Assays.
- Author
-
Scheele S, Geiger JA, DeRocher AE, Choi R, Smith TR, Hulverson MA, Vidadala RSR, Barrett LK, Maly DJ, Merritt EA, Ojo KK, Van Voorhis WC, and Parsons M
- Subjects
- Animals, Drug Resistance genetics, Focal Adhesion Kinase 2 genetics, Humans, Protozoan Proteins antagonists & inhibitors, Protozoan Proteins genetics, Toxoplasma genetics, Focal Adhesion Kinase 2 antagonists & inhibitors, Naphthalenes pharmacology, Piperidines pharmacology, Protein Kinase Inhibitors pharmacology, Pyrazoles pharmacology, Toxoplasma drug effects, Toxoplasmosis drug therapy
- Abstract
In Toxoplasma gondii , calcium-dependent protein kinase 1 (CDPK1) is an essential protein kinase required for invasion of host cells. We have developed several hundred CDPK1 inhibitors, many of which block invasion. Inhibitors with similar 50% inhibitory concentrations (IC
50 s) were tested in thermal shift assays for their ability to stabilize CDPK1 in cell lysates, in intact cells, or in purified form. Compounds that inhibited parasite growth stabilized CDPK1 in all assays. In contrast, two compounds that showed poor growth inhibition stabilized CDPK1 in lysates but not in cells. Thus, cellular exclusion could explain exceptions in the correlation between the action on the target and cellular activity. We used thermal shift assays to examine CDPK1 in two clones that were independently selected by growth in the CDPK1 inhibitor RM-1-132 and that had increased 50% effective concentrations (EC50 s) for the compound. The A and C clones had distinct point mutations in the CDPK1 kinase domain, H201Q and L96P, respectively, residues that lie near one another in the inactive isoform. Purified mutant proteins showed RM-1-132 IC50 s and thermal shifts similar to those shown by wild-type CDPK1. Reduced inhibitor stabilization (and a presumed reduced interaction) was observed only in cellular thermal shift assays. This highlights the utility of cellular thermal shift assays in demonstrating that resistance involves reduced on-target engagement (even if biochemical assays suggest otherwise). Indeed, similar EC50 s were observed upon overexpression of the mutant proteins, as in the corresponding drug-selected parasites, although high levels of CDPK1(H201Q) only modestly increased resistance compared to that achieved with high levels of wild-type enzyme., (Copyright © 2018 American Society for Microbiology.)- Published
- 2018
- Full Text
- View/download PDF
43. 7 H-Pyrrolo[2,3- d]pyrimidin-4-amine-Based Inhibitors of Calcium-Dependent Protein Kinase 1 Have Distinct Inhibitory and Oral Pharmacokinetic Characteristics Compared with 1 H-Pyrazolo[3,4- d]pyrimidin-4-amine-Based Inhibitors.
- Author
-
Vidadala RSR, Golkowski M, Hulverson MA, Choi R, McCloskey MC, Whitman GR, Huang W, Arnold SLM, Barrett LK, Fan E, Merritt EA, Van Voorhis WC, Ojo KK, and Maly DJ
- Subjects
- Animals, Antiprotozoal Agents chemical synthesis, Antiprotozoal Agents pharmacokinetics, Antiprotozoal Agents toxicity, Cell Line, Cell Survival drug effects, Cryptosporidium parvum drug effects, Cryptosporidium parvum growth & development, Enzyme Inhibitors chemical synthesis, Enzyme Inhibitors pharmacokinetics, Enzyme Inhibitors toxicity, Humans, Inhibitory Concentration 50, Mice, Inbred BALB C, Molecular Structure, Pyrimidines chemical synthesis, Pyrimidines pharmacokinetics, Pyrimidines toxicity, Pyrroles chemical synthesis, Pyrroles pharmacokinetics, Pyrroles toxicity, Structure-Activity Relationship, Antiprotozoal Agents pharmacology, Cryptosporidium parvum enzymology, Enzyme Inhibitors pharmacology, Protein Kinases metabolism, Pyrimidines pharmacology, Pyrroles pharmacology
- Abstract
Selective inhibitors of Cryptosporidium calcium-dependent protein kinase 1 ( CpCDPK1) based on the 1 H-pyrazolo[3,4- d]pyrimidin-4-amine (pyrazolopyrimidine, PP) scaffold are effective in both in vitro and in vivo models of cryptosporidiosis. However, the search for distinct safety and pharmacokinetic (PK) properties has motivated our exploration of alternative scaffolds. Here, we describe a series of 7 H-pyrrolo[2,3- d]pyrimidin-4-amine (pyrrolopyrimidine, PrP)-based analogs of PP CpCDPK1 inhibitors. Most of the PrP-based inhibitors described potently inhibit the CpCDPK1 enzyme, demonstrate no toxicity against mammalian cells, and block proliferation of the C. parvum parasite in the low micromolar range. Interestingly, certain substituents that show reduced CpCDPK1 potency when displayed from a PP scaffold provided notably enhanced efficacy in the context of a PrP scaffold. PK studies on these paired compounds show that some PrP analogs have distinct physiochemical properties compared with their PP counterparts. These results demonstrate that inhibitors based on a PrP scaffold are distinct therapeutic alternatives to previously developed PP inhibitors.
- Published
- 2018
- Full Text
- View/download PDF
44. Rheostatic Control of Cas9-Mediated DNA Double Strand Break (DSB) Generation and Genome Editing.
- Author
-
Rose JC, Stephany JJ, Wei CT, Fowler DM, and Maly DJ
- Subjects
- Benzothiazoles pharmacology, Gene Editing, HEK293 Cells, Humans, INDEL Mutation genetics, Isoquinolines pharmacology, Polymerase Chain Reaction methods, Streptococcus pyogenes enzymology, bcl-X Protein antagonists & inhibitors, CRISPR-Associated Protein 9 metabolism, CRISPR-Associated Proteins metabolism, CRISPR-Cas Systems genetics, DNA genetics, DNA Breaks, Double-Stranded
- Abstract
We recently reported two novel tools for precisely controlling and quantifying Cas9 activity: a chemically inducible Cas9 variant (ciCas9) that can be rapidly activated by small molecules and a ddPCR assay for time-resolved measurement of DNA double strand breaks (DSB-ddPCR). Here, we further demonstrate the potential of ciCas9 to function as a tunable rheostat for Cas9 function. We show that a new highly potent and selective small molecule activator paired with a more tightly regulated ciCas9 variant expands the range of accessible Cas9 activity levels. We subsequently demonstrate that ciCas9 activity levels can be dose-dependently tuned with a small molecule activator, facilitating rheostatic time-course experiments. These studies provide the first insight into how Cas9-mediated DSB levels correlate with overall editing efficiency. Thus, we demonstrate that ciCas9 and our DSB-ddPCR assay permit the time-resolved study of Cas9 DSB generation and genome editing kinetics at a wide range of Cas9 activity levels.
- Published
- 2018
- Full Text
- View/download PDF
45. In vitro growth inhibition of Theileria equi by bumped kinase inhibitors.
- Author
-
Gimenez F, Hines SA, Evanoff R, Ojo KK, Van Voorhis WC, Maly DJ, Vidadala RSR, and Mealey RH
- Subjects
- Animals, Antiprotozoal Agents therapeutic use, Babesiosis drug therapy, Babesiosis parasitology, Cattle, Horse Diseases drug therapy, Horse Diseases parasitology, Horses parasitology, Humans, Imidocarb analogs & derivatives, Imidocarb pharmacology, Protein Kinase Inhibitors therapeutic use, Theileria growth & development, Theileriasis drug therapy, Theileriasis epidemiology, Antiprotozoal Agents pharmacology, Protein Kinase Inhibitors pharmacology, Theileria drug effects
- Abstract
Introduction: Theileria equi, an etiologic agent of equine piroplasmosis, is a tick-transmitted hemoprotozoan of the phylum Apicomplexa. Recent outbreaks of piroplasmosis in the United States have renewed interest in safe and effective treatment options. Although imidocarb dipropionate (IMD) is the drug of choice for clearance of T. equi, adverse reactions and recently documented resistance support the need for alternative therapeutic strategies. The recently described bumped kinase inhibitors (BKIs) are a new class of compounds that could potentially be used as safe and effective alternatives to IMD. In an initial effort to evaluate this potential, herein we determined the T. equi growth inhibitory activity of 11 BKIs relative to that of IMD and the previously tested BKI 1294. Because some BKIs have known human ether-à-go-go related gene (hERG) channel activity, we also assessed the hERG activity of each compound with the goal to identify those with the highest potency against T. equi coupled with the lowest potential for cardiotoxicity., Results: Six BKIs inhibited T. equi growth in vitro, including the previously evaluated BKI 1294 which was used as a positive control. All six compounds were significantly less potent (higher 50% effective concentration (EC
50 )) than IMD. Two of those compounds were more potent than BKI 1294 control but had similar hERG activity. Although the remaining three compounds had similar to lower potency than BKI 1294, hERG EC50 was higher for three of them (BKI 1735, BKI 1369 and BKI 1318)., Conclusions: The BKI compounds evaluated in this study inhibited T. equi in vitro and had diverse hERG activity. Based on these considerations, three compounds would be suitable for further evaluation. While these results provide a foundation for future work, in vivo pharmacokinetic, pharmacodynamics, and safety studies are needed before BKI compounds can be recommended for clinical use in T. equi infected horses., (Copyright © 2018 Elsevier B.V. All rights reserved.)- Published
- 2018
- Full Text
- View/download PDF
46. Kinome chemoproteomics characterization of pyrrolo[3,4-c]pyrazoles as potent and selective inhibitors of glycogen synthase kinase 3.
- Author
-
Golkowski M, Perera GK, Vidadala VN, Ojo KK, Van Voorhis WC, Maly DJ, and Ong SE
- Abstract
Glycogen synthase kinase 3 has evolutionarily conserved roles in cell signaling and metabolism and is a recognized drug target in neurological pathologies, most prominently bipolar disorder. More recently it has been suggested that GSK3 may be a target for the treatment of trypanosomatid parasite infections, e.g. with T. brucei, due to the lethal phenotype observed in parasite GSK3 short RNAi knockdown experiments. Here we investigated the kinome selectivity of a library of pyrrolo[3,4-c]pyrazol inhibitors that were developed against T. brucei GSK3 but that also interact with the human orthologue and other protein kinases. We applied label-free MS-based kinome chemoproteomics profiling with kinobeads to obtain the selectivity profiles of all 39 library members against 217 human protein and lipid kinases. This allowed us to study the structure-activity relationship of the library members as well as the chemical genetic relationships between kinase targets. As a result, we identified a novel and highly selective HsGSK3 inhibitor containing a 2-chloroaniline-substituted squaric acid amide pharmacophore that confers low nanomolar (IC
50 = 2.8 nM) and sub-micromolar potency against purified and cellular HsGSK3. The inhibitor will be useful as a new lead for GSK3 inhibitor development and as a chemical genetic probe to study roles of GSK3 in cell signaling.- Published
- 2018
- Full Text
- View/download PDF
47. Advances in bumped kinase inhibitors for human and animal therapy for cryptosporidiosis.
- Author
-
Hulverson MA, Choi R, Arnold SLM, Schaefer DA, Hemphill A, McCloskey MC, Betzer DP, Müller J, Vidadala RSR, Whitman GR, Rivas KL, Barrett LK, Hackman RC, Love MS, McNamara CW, Shaughnessy TK, Kondratiuk A, Kurnick M, Banfor PN, Lynch JJ, Freiberg GM, Kempf DJ, Maly DJ, Riggs MW, Ojo KK, and Van Voorhis WC
- Subjects
- Administration, Oral, Animals, Animals, Newborn, Cattle, Disease Models, Animal, Dose-Response Relationship, Drug, Female, Heart drug effects, Humans, Inhibitory Concentration 50, Interferon-gamma genetics, Male, Mice, Mice, Inbred BALB C, Mice, Knockout, Mutagenicity Tests, Pregnancy, Protein Binding, Protein Kinase Inhibitors administration & dosage, Protein Kinase Inhibitors blood, Protein Kinase Inhibitors toxicity, Safety, Cryptosporidiosis drug therapy, Cryptosporidium parvum drug effects, Protein Kinase Inhibitors therapeutic use
- Abstract
Improvements have been made to the safety and efficacy of bumped kinase inhibitors, and they are advancing toward human and animal use for treatment of cryptosporidiosis. As the understanding of bumped kinase inhibitor pharmacodynamics for cryptosporidiosis therapy has increased, it has become clear that better compounds for efficacy do not necessarily require substantial systemic exposure. We now have a bumped kinase inhibitor with reduced systemic exposure, acceptable safety parameters, and efficacy in both the mouse and newborn calf models of cryptosporidiosis. Potential cardiotoxicity is the limiting safety parameter to monitor for this bumped kinase inhibitor. This compound is a promising pre-clinical lead for cryptosporidiosis therapy in animals and humans., (Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
48. In vitro efficacy of bumped kinase inhibitors against Besnoitia besnoiti tachyzoites.
- Author
-
Jiménez-Meléndez A, Ojo KK, Wallace AM, Smith TR, Hemphill A, Balmer V, Regidor-Cerrillo J, Ortega-Mora LM, Hehl AB, Fan E, Maly DJ, Van Voorhis WC, and Álvarez-García G
- Subjects
- Amino Acid Sequence, Base Sequence, Cell Line, Cloning, Molecular, DNA, Protozoan chemistry, DNA, Protozoan isolation & purification, Dose-Response Relationship, Drug, Drug Evaluation, Preclinical, Fibroblasts cytology, Fibroblasts parasitology, Fluorescent Antibody Technique, Humans, Male, Microscopy, Electron, Transmission, Protein Kinases chemistry, Protein Kinases drug effects, Protein Kinases genetics, Real-Time Polymerase Chain Reaction, Sarcocystidae genetics, Sarcocystidae growth & development, Sarcocystidae ultrastructure, Serial Passage, Protein Kinase Inhibitors pharmacology, Protein Kinases isolation & purification, Sarcocystidae drug effects
- Abstract
Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a chronic and debilitating disease that causes systemic and skin manifestations and sterility in bulls. Neither treatments nor vaccines are currently available. In the search for therapeutic candidates, calcium-dependent protein kinases have arisen as promising drug targets in other apicomplexans (e.g. Neospora caninum, Toxoplasma gondii, Plasmodium spp. and Eimeria spp.) and are effectively targeted by bumped kinase inhibitors. In this study, we identified and cloned the gene coding for BbCDPK1. The impact of a library of nine bumped kinase inhibitor analogues on the activity of recombinant BbCDPK1 was assessed by luciferase assay. Afterwards, those were further screened for efficacy against Besnoitiabesnoiti tachyzoites grown in Marc-145 cells. Primary tests at 5µM revealed that eight compounds exhibited more than 90% inhibition of invasion and proliferation. The compounds BKI 1294, 1517, 1553 and 1571 were further characterised, and EC
99 (1294: 2.38µM; 1517: 2.20µM; 1553: 3.34µM; 1571: 2.78µM) were determined by quantitative real-time polymerase chain reaction in 3-day proliferation assays. Exposure of infected cultures with EC99 concentrations of these drugs for up to 48h was not parasiticidal. The lack of parasiticidal action was confirmed by transmission electron microscopy, which showed that bumped kinase inhibitor treatment interfered with cell cycle regulation and non-disjunction of tachyzoites, resulting in the formation of large multi-nucleated complexes which co-existed with viable parasites within the parasitophorous vacuole. However, it is possible that, in the face of an active immune response, parasite clearance may occur. In summary, bumped kinase inhibitors may be effective drug candidates to control Besnoitiabesnoiti infection. Further in vivo experiments should be planned, as attainment and maintenance of therapeutic blood plasma levels in calves, without toxicity, has been demonstrated for BKIs 1294, 1517 and 1553., (Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)- Published
- 2017
- Full Text
- View/download PDF
49. Rapidly inducible Cas9 and DSB-ddPCR to probe editing kinetics.
- Author
-
Rose JC, Stephany JJ, Valente WJ, Trevillian BM, Dang HV, Bielas JH, Maly DJ, and Fowler DM
- Subjects
- Kinetics, Caspase 9 genetics, DNA Breaks, Double-Stranded, DNA Probes genetics, Gene Editing methods, Molecular Probe Techniques, Polymerase Chain Reaction methods
- Abstract
We developed a chemically inducible Cas9 (ciCas9) and a droplet digital PCR assay for double-strand breaks (DSB-ddPCR) to investigate the kinetics of Cas9-mediated generation and repair of DSBs in cells. ciCas9 is a rapidly activated, single-component Cas9 variant engineered by replacing the protein's REC2 domain with the BCL-xL protein and fusing an interacting BH3 peptide to the C terminus. ciCas9 can be tunably activated by a compound that disrupts the BCL-xL-BH3 interaction within minutes. DSB-ddPCR demonstrates time-resolved, highly quantitative, and targeted measurement of DSBs. Combining these tools facilitated an unprecedented exploration of the kinetics of Cas9-mediated DNA cleavage and repair. We find that sgRNAs targeting different sites generally induce cleavage within minutes and repair within 1 or 2 h. However, we observe distinct kinetic profiles, even for proximal sites, and this suggests that target sequence and chromatin state modulate cleavage and repair kinetics.
- Published
- 2017
- Full Text
- View/download PDF
50. Extended-spectrum antiprotozoal bumped kinase inhibitors: A review.
- Author
-
Van Voorhis WC, Doggett JS, Parsons M, Hulverson MA, Choi R, Arnold SLM, Riggs MW, Hemphill A, Howe DK, Mealey RH, Lau AOT, Merritt EA, Maly DJ, Fan E, and Ojo KK
- Subjects
- Animals, Antiprotozoal Agents therapeutic use, Apicomplexa enzymology, Benzimidazoles chemistry, Humans, Imidazoles chemistry, Protein Kinase Inhibitors therapeutic use, Protozoan Infections prevention & control, Pyridines chemistry, Antiprotozoal Agents pharmacology, Apicomplexa drug effects, Protein Kinase Inhibitors pharmacology, Protein-Tyrosine Kinases antagonists & inhibitors, Protozoan Infections drug therapy
- Abstract
Many life-cycle processes in parasites are regulated by protein phosphorylation. Hence, disruption of essential protein kinase function has been explored for therapy of parasitic diseases. However, the difficulty of inhibiting parasite protein kinases to the exclusion of host orthologues poses a practical challenge. A possible path around this difficulty is the use of bumped kinase inhibitors for targeting calcium-dependent protein kinases that contain atypically small gatekeeper residues and are crucial for pathogenic apicomplexan parasites' survival and proliferation. In this article, we review efficacy against the kinase target, parasite growth in vitro, and in animal infection models, as well as the relevant pharmacokinetic and safety parameters of bumped kinase inhibitors., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.