Neospora caninum: Differential Proteome of Multinucleated Complexes Induced by the Bumped Kinase Inhibitor BKI-1294

Background: the apicomplexan parasite Neospora caninum causes important reproductive problems in farm animals, most notably in cattle. After infection via oocysts or tissue cysts, rapidly dividing tachyzoites infect various tissues and organs, and in immunocompetent hosts, they differentiate into slowly dividing bradyzoites, which form tissue cysts and constitute a resting stage persisting within infected tissues. Bumped kinase inhibitors (BKIs) of calcium dependent protein kinase 1 are promising drug candidates for the treatment of Neospora infections. BKI-1294 exposure of cell cultures infected with N. caninum tachyzoites results in the formation of massive multinucleated complexes (MNCs) containing numerous newly formed zoites, which remain viable for extended periods of time under drug pressure in vitro. MNC and tachyzoites exhibit considerable antigenic and structural differences. Methods: Using shotgun mass spectrometry, we compared the proteomes of tachyzoites to BKI-1294 induced MNCs, and analyzed the mRNA expression levels of selected genes in both stages. Results: More than half of the identified proteins are downregulated in MNCs as compared to tachyzoites. Only 12 proteins are upregulated, the majority of them containing SAG1 related sequence (SRS) domains, and some also known to be expressed in bradyzoites Conclusions: MNCs exhibit a proteome different from tachyzoites, share some bradyzoite-like features, but may constitute a third stage, which remains viable and ensures survival under adverse conditions such as drug pressure. We propose the term “baryzoites” for this stage (from Greek βαρυσ = massive, bulky, heavy, inert).

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Neospora caninum: Structure and Fate of Multinucleated Complexes Induced by the Bumped Kinase Inhibitor BKI-1294

Pathogens ◽

10.3390/pathogens9050382 ◽

2020 ◽

Vol 9 (5) ◽

pp. 382 ◽

Cited By ~ 2

Author(s):

Pablo Winzer ◽

Nicoleta Anghel ◽

Dennis Imhof ◽

Vreni Balmer ◽

Luis-Miguel Ortega-Mora ◽

...

Keyword(s):

Drug Exposure ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Neospora Caninum ◽

Farm Animals ◽

In Vivo Studies ◽

Drug Pressure ◽

Drug Removal

Background: Bumped kinase inhibitors (BKIs) are potential drugs for neosporosis treatment in farm animals. BKI-1294 exposure results in the formation of multinucleated complexes (MNCs), which remain viable in vitro under constant drug pressure. We investigated the formation of BKI-1294 induced MNCs, the re-emergence of viable tachyzoites following drug removal, and the localization of CDPK1, the molecular target of BKIs. Methods: N. caninum tachyzoites and MNCs were studied by TEM and immunofluorescence using antibodies directed against CDPK1, and against NcSAG1 and IMC1 as markers for tachyzoites and newly formed zoites, respectively. Results: After six days of drug exposure, MNCs lacked SAG1 surface expression but remained intracellular, and formed numerous zoites incapable of disjoining from each other. Following drug removal, proliferation continued, and zoites lacking NcSAG1 emerged from the periphery of these complexes, forming infective tachyzoites after 10 days. In intracellular tachyzoites, CDPK1 was evenly distributed but shifted towards the apical part once parasites were extracellular. This shift was not affected by BKI-1294. Conclusions: CDPK1 has a dynamic distribution depending on whether parasites are located within a host cell or outside. During MNC-to-tachyzoite reconversion newly formed tachyzoites are generated directly from MNCs through zoites of unknown surface antigen composition. Further in vivo studies are needed to determine if MNCs could lead to a persistent reservoir of infection after BKI treatment.

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Two Novel Calcium-Dependent Protein Kinase 1 Inhibitors Interfere with Vertical Transmission in Mice Infected with Neospora caninum Tachyzoites

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02324-16 ◽

2017 ◽

Vol 61 (4) ◽

Cited By ~ 11

Author(s):

Joachim Müller ◽

Adriana Aguado-Martínez ◽

Vreni Balmer ◽

Dustin J. Maly ◽

Erkang Fan ◽

...

Keyword(s):

Vertical Transmission ◽

Kinase Inhibitors ◽

Prolonged Exposure ◽

Neospora Caninum ◽

Morphological Changes ◽

Parasite Load ◽

Content Type ◽

Calcium Dependent ◽

Dependent Protein

ABSTRACT We present the effects of two novel bumped kinase inhibitors, BKI-1517 and BKI-1553, against Neospora caninum tachyzoites in vitro and in experimentally infected pregnant mice. These compounds inhibited tachyzoite proliferation of a transgenic beta-galactosidase reporter strain cultured in human foreskin fibroblasts with 50% inhibitory concentrations (IC50s) of 0.05 ± 0.03 and 0.18 ± 0.03 μM, respectively. As assessed by an alamarBlue assay, fibroblast IC50s were above 20 μM; however, morphological changes occurred in cultures treated with >5 μM BKI-1517 after prolonged exposure (>6 days). Treatment of intracellular tachyzoites with 5 μM BKI-1553 for 6 days inhibited endodyogeny by interfering with the separation of newly formed zoites from a larger multinucleated parasite mass. In contrast, parasites treated with 5 μM BKI-1517 did not form large complexes and showed much more evidence of cell death. However, after a treatment duration of 10 days in vitro, both compounds failed to completely prevent the regrowth of parasites from culture. BALB/c mice experimentally infected with N. caninum Spain7 (Nc-Spain7) and then treated during 6 days with BKI-1517 or BKI-1553 at different dosages showed a significant reduction of the cerebral parasite load. However, fertility was impaired by BKI-1517 when applied at 50 mg/kg of body weight/day. At 20 mg/kg/day, BKI-1517 significantly inhibited the vertical transmission of N. caninum to pups and increased the rate of survival of offspring. BKI-1553 was less detrimental to fertility and also provided significant but clearly less pronounced protection of dams and offspring. These results demonstrate that, when judiciously applied, this compound class protects offspring from vertical transmission and disease.

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The gatekeeper residue and beyond: homologous calcium-dependent protein kinases as drug development targets for veterinarian Apicomplexa parasites

Parasitology ◽

10.1017/s0031182014000857 ◽

2014 ◽

Vol 141 (11) ◽

pp. 1499-1509 ◽

Cited By ~ 34

Author(s):

KATELYN R. KEYLOUN ◽

MOLLY C. REID ◽

RYAN CHOI ◽

YIFAN SONG ◽

ANNA M. W. FOX ◽

...

Keyword(s):

Amino Acid ◽

Protein Kinases ◽

Kinase Inhibitors ◽

Neospora Caninum ◽

Babesia Bovis ◽

Atp Binding ◽

Calcium Dependent ◽

Binding Cavity ◽

Dependent Protein ◽

Calcium Dependent Protein Kinases

SUMMARYSpecific roles of individual CDPKs vary, but in general they mediate essential biological functions necessary for parasite survival. A comparative analysis of the structure-activity relationships (SAR) of Neospora caninum, Eimeria tenella and Babesia bovis calcium-dependent protein kinases (CDPKs) together with those of Plasmodium falciparum, Cryptosporidium parvum and Toxoplasma gondii was performed by screening against 333 bumped kinase inhibitors (BKIs). Structural modelling and experimental data revealed that residues other than the gatekeeper influence compound–protein interactions resulting in distinct sensitivity profiles. We subsequently defined potential amino-acid structural influences within the ATP-binding cavity for each orthologue necessary for consideration in the development of broad-spectrum apicomplexan CDPK inhibitors. Although the BKI library was developed for specific inhibition of glycine gatekeeper CDPKs combined with low inhibition of threonine gatekeeper human SRC kinase, some library compounds exhibit activity against serine- or threonine-containing CDPKs. Divergent BKI sensitivity of CDPK homologues could be explained on the basis of differences in the size and orientation of the hydrophobic pocket and specific variation at other amino-acid positions within the ATP-binding cavity. In particular, BbCDPK4 and PfCDPK1 are sensitive to a larger fraction of compounds than EtCDPK1 despite the presence of a threonine gatekeeper in all three CDPKs.

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Bumped Kinase Inhibitor 1294 Treats Established Toxoplasma gondii Infection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01823-13 ◽

2014 ◽

Vol 58 (6) ◽

pp. 3547-3549 ◽

Cited By ~ 42

Author(s):

J. Stone Doggett ◽

Kayode K. Ojo ◽

Erkang Fan ◽

Dustin J. Maly ◽

Wesley C. Van Voorhis

Keyword(s):

Toxoplasma Gondii ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Content Type ◽

Calcium Dependent ◽

Dependent Protein ◽

Orally Bioavailable ◽

Toxoplasma Gondii Infection ◽

Bumped Kinase Inhibitor

ABSTRACTToxoplasma gondiiis a unicellular parasite that causes severe brain and eye disease. Current drugs forT. gondiiare limited by toxicity. Bumped kinase inhibitors (BKIs) selectively inhibit calcium-dependent protein kinases of the apicomplexan pathogensT. gondii, cryptosporidia, and plasmodia. A lead anti-ToxoplasmaBKI, 1294, has been developed to be metabolically stable and orally bioavailable. Herein, we demonstrate the oral efficacy of 1294 against toxoplasmosisin vivo.

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Antineoplastic kinase inhibitors: a new class of potent anti-amoebic compounds Antineoplastic kinase inhibitors against Entamoeba histolytica

10.1101/2020.05.28.120923 ◽

2020 ◽

Author(s):

Conall Sauvey ◽

Gretchen Ehrenkaufer ◽

Da Shi ◽

Anjan Debnath ◽

Ruben Abagyan

Keyword(s):

Entamoeba Histolytica ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Model Organism ◽

Protozoan Parasite ◽

Drug Candidates ◽

Life Threatening ◽

The 1960S ◽

Approved Drugs

AbstractEntamoeba histolytica is a protozoan parasite which infects approximately 50 million people worldwide, resulting in an estimated 70,000 deaths every year. Since the 1960s E. histolytica infection has been successfully treated with metronidazole. However, drawbacks to metronidazole therapy exist, including adverse effects, a long treatment course, and the need for an additional drug to prevent cyst-mediated transmission. E. histolytica possesses a kinome with approximately 300 - 400 members, some of which have been previously studied as potential targets for the development of amoebicidal drug candidates. However, while these efforts have uncovered novel potent inhibitors of E. histolytica kinases, none have resulted in approved drugs. In this study we took the alternative approach of testing a set of twelve previously FDA-approved antineoplastic kinase inhibitors against E. histolytica trophozoites in vitro. This resulted in the identification of dasatinib, bosutinib, and ibrutinib as amoebicidal agents at low-micromolar concentrations. Next, we utilized a recently developed computational tool to identify twelve additional drugs with human protein target profiles similar to the three initial hits. Testing of these additional twelve drugs led to the identification of ponatinib, neratinib, and olmutinib were identified as highly potent, with EC50 values in the sub-micromolar range. All of these six drugs were found to kill E. histolytica trophozoites as rapidly as metronidazole. Furthermore, ibrutinib was found to kill the transmissible cyst stage of the model organism E. invadens. Ibrutinib thus possesses both amoebicidal and cysticidal properties, in contrast to all drugs used in the current therapeutic strategy. These findings together reveal antineoplastic kinase inhibitors as a highly promising class of potent drugs against this widespread and devastating disease.Author SummaryEvery year, nearly a hundred thousand people worldwide die from infection by the intestinal parasite Entamoeba histolytica, despite the widespread availability of metronidazole as a treatment. Here we report that six anticancer drugs of the kinase inhibitor class possess potent anti-amoebic properties, with one of them killing both actively dividing parasite and its transmissible cysts. These anticancer kinase inhibitors, including the dual-purpose drug with both amoebicidal and cysticidal activities may be used to treat amoebiasis, especially in cancer patients or in life-threatening brain- and liver-infecting forms of the disease.

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Targeting USP47 overcomes tyrosine kinase inhibitor resistance and eradicates leukemia stem/progenitor cells in chronic myelogenous leukemia

Nature Communications ◽

10.1038/s41467-020-20259-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Hu Lei ◽

Han-Zhang Xu ◽

Hui-Zhuang Shan ◽

Meng Liu ◽

Ying Lu ◽

...

Keyword(s):

Tyrosine Kinase ◽

Progenitor Cells ◽

Chronic Myelogenous Leukemia ◽

Drug Targets ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Myelogenous Leukemia ◽

Tki Resistance

AbstractIdentifying novel drug targets to overcome resistance to tyrosine kinase inhibitors (TKIs) and eradicating leukemia stem/progenitor cells are required for the treatment of chronic myelogenous leukemia (CML). Here, we show that ubiquitin-specific peptidase 47 (USP47) is a potential target to overcome TKI resistance. Functional analysis shows that USP47 knockdown represses proliferation of CML cells sensitive or resistant to imatinib in vitro and in vivo. The knockout of Usp47 significantly inhibits BCR-ABL and BCR-ABLT315I-induced CML in mice with the reduction of Lin−Sca1+c-Kit+ CML stem/progenitor cells. Mechanistic studies show that stabilizing Y-box binding protein 1 contributes to USP47-mediated DNA damage repair in CML cells. Inhibiting USP47 by P22077 exerts cytotoxicity to CML cells with or without TKI resistance in vitro and in vivo. Moreover, P22077 eliminates leukemia stem/progenitor cells in CML mice. Together, targeting USP47 is a promising strategy to overcome TKI resistance and eradicate leukemia stem/progenitor cells in CML.

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Regulation of PDGFR-α in rat pulmonary myofibroblasts by staurosporine

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2001.280.2.l354 ◽

2001 ◽

Vol 280 (2) ◽

pp. L354-L362 ◽

Cited By ~ 7

Author(s):

Pamela M. Lindroos ◽

Yi-Zhe Wang ◽

Annette B. Rice ◽

James C. Bonner

Keyword(s):

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Protein A ◽

Nuclear Factor Κb ◽

Mitogen Activated Protein Kinase ◽

Specific Gene ◽

Autocrine Loop ◽

P38 Inhibitor ◽

Mitogen Activated Protein

Upregulation of the platelet-derived growth factor (PDGF) receptor-α (PDGFR-α) is a mechanism of myofibroblast hyperplasia during pulmonary fibrosis. We previously identified interleukin (IL)-1β as a major inducer of the PDGFR-α in rat pulmonary myofibroblasts in vitro. In this study, we report that staurosporine, a broad-spectrum kinase inhibitor, upregulates PDGFR-α gene expression and protein. A variety of other kinase inhibitors did not induce PDGFR-α expression. Staurosporine did not act via an IL-1β autocrine loop because the IL-1 receptor antagonist protein did not block staurosporine-induced PDGFR-α expression. Furthermore, staurosporine did not activate a variety of signaling molecules that were activated by IL-1β, including nuclear factor-κB, extracellular signal-regulated kinase, and c-Jun NH2-terminal kinase. However, both staurosporine- and IL-1β-induced phosphorylation of p38 mitogen-activated protein kinase and upregulation of PDGFR-α by these two agents was inhibited by the p38 inhibitor SB-203580. Finally, staurosporine inhibited basal and PDGF-stimulated mitogenesis over the same concentration range that induced PDGFR-α expression. Collectively, these data demonstrate that staurosporine is a useful tool for elucidating the signaling mechanisms that regulate PDGFR expression in lung connective tissue cells and possibly for evaluating the role of the PDGFR-α as a growth arrest-specific gene.

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Identification of Hsp90 inhibitors as potential drugs for the treatment of TSC1/TSC2 deficient cancer

10.1101/2021.02.26.433022 ◽

2021 ◽

Author(s):

Evelyn M. Mrozek ◽

Vineeta Bajaj ◽

Yanan Guo ◽

Izabela Malinowska ◽

Jianming Zhang ◽

...

Keyword(s):

Cell Lines ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Xenograft Model ◽

Growth Delay ◽

Hsp90 Inhibitors ◽

Null Cell ◽

Standard Dosage

Inactivating mutations in either TSC1 or TSC2 cause Tuberous Sclerosis Complex, an autosomal dominant disorder, characterized by multi-system tumor and hamartoma development. Mutation and loss of function of TSC1 and/or TSC2 also occur in a variety of sporadic cancers, and rapamycin and related drugs show highly variable treatment benefit in patients with such cancers. The TSC1 and TSC2 proteins function in a complex that inhibits mTORC1, a key regulator of cell growth, which acts to enhance anabolic biosynthetic pathways. In this study, we identified and validated five cancer cell lines with TSC1 or TSC2 mutations and performed a kinase inhibitor drug screen with 197 compounds. The five cell lines were sensitive to several mTOR inhibitors, and cell cycle kinase and HSP90 kinase inhibitors. The IC50 for Torin1 and INK128, both mTOR kinase inhibitors, was significantly increased in three TSC2 null cell lines in which TSC2 expression was restored. Rapamycin was significantly more effective than either INK128 or ganetespib (an HSP90 inhibitor) in reducing the growth of TSC2 null SNU-398 cells in a xenograft model. Combination ganetespib-rapamycin showed no significant enhancement of growth suppression over rapamycin. Hence, although HSP90 inhibitors show strong inhibition of TSC1/TSC2 null cell line growth in vitro, ganetespib showed little benefit at standard dosage in vivo. In contrast, rapamycin which showed very modest growth inhibition in vitro was the best agent for in vivo treatment, but did not cause tumor regression, only growth delay.

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Covalent inhibitors of EGFR family protein kinases induce degradation of human Tribbles 2 (TRIB2) pseudokinase in cancer cells

Science Signaling ◽

10.1126/scisignal.aat7951 ◽

2018 ◽

Vol 11 (549) ◽

pp. eaat7951 ◽

Cited By ~ 23

Author(s):

Daniel M. Foulkes ◽

Dominic P. Byrne ◽

Wayland Yeung ◽

Safal Shrestha ◽

Fiona P. Bailey ◽

...

Keyword(s):

Cancer Cells ◽

Small Molecule ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Growth Factor Receptor ◽

Drug Repurposing ◽

Protein Kinase Inhibitors ◽

Cellular Mechanisms ◽

Cellular Analysis

A major challenge associated with biochemical and cellular analysis of pseudokinases is a lack of target-validated small-molecule compounds with which to probe function. Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the canonical AKT signaling module. There is substantial evidence that human TRIB2 promotes survival and drug resistance in solid tumors and blood cancers and therefore is of interest as a therapeutic target. The unusual TRIB2 pseudokinase domain contains a unique cysteine-rich C-helix and interacts with a conserved peptide motif in its own carboxyl-terminal tail, which also supports its interaction with E3 ubiquitin ligases. We found that TRIB2 is a target of previously described small-molecule protein kinase inhibitors, which were originally designed to inhibit the canonical kinase domains of epidermal growth factor receptor tyrosine kinase family members. Using a thermal shift assay, we discovered TRIB2-binding compounds within the Published Kinase Inhibitor Set (PKIS) and used a drug repurposing approach to classify compounds that either stabilized or destabilized TRIB2 in vitro. TRIB2 destabilizing agents, including the covalent drug afatinib, led to rapid TRIB2 degradation in human AML cancer cells, eliciting tractable effects on signaling and survival. Our data reveal new drug leads for the development of TRIB2-degrading compounds, which will also be invaluable for unraveling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule–induced protein down-regulation through drug “off-targets” might be relevant for other inhibitors that serendipitously target pseudokinases.

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New tools for carbohydrate sulfation analysis: heparan sulfate 2-O-sulfotransferase (HS2ST) is a target for small-molecule protein kinase inhibitors

Biochemical Journal ◽

10.1042/bcj20180265 ◽

2018 ◽

Vol 475 (15) ◽

pp. 2417-2433 ◽

Cited By ~ 8

Author(s):

Dominic P. Byrne ◽

Yong Li ◽

Krithika Ramakrishnan ◽

Igor L. Barsukov ◽

Edwin A. Yates ◽

...

Keyword(s):

Protein Kinase ◽

Heparan Sulfate ◽

Small Molecule ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Protein Kinase Inhibitors ◽

Fluorescent Substrate ◽

Raf Kinase ◽

Shift Analysis

Sulfation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulfate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulfotransferases, including HS 2-O-sulfotransferase (HS2ST), which transfers sulfate from the cofactor PAPS (3′-phosphoadenosine 5′-phosphosulfate) to the 2-O position of α-l-iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulfation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In the present paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalysed oligosaccharide sulfation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set, to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl protein sulfotranferases are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulfation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.

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