Development of an Antivirulence Drug againstStreptococcus mutans: Repression of Biofilm Formation, Acid Tolerance, and Competence by a Histidine Kinase Inhibitor, Walkmycin C

ABSTRACTTwo-component signal transduction systems (TCSs) in prokaryotes often regulate gene clusters that induce pathogenicity, and thus they have frequently been proposed as potential drug targets for attenuating the virulence of pathogens. The pathogenic potential ofStreptococcus mutans, the major etiological pathogen of dental caries, is also regulated by its TCSs. The object of this study was to evaluate the effect of a histidine kinase (HK) inhibitor against two major virulence factors ofS. mutans: biofilm formation and acid tolerance. Walkmycin C (WKM C), an HK inhibitor isolated from the screening of inhibitors against WalK HK inBacillus subtilis, inhibited thein vitroautophosphorylation activity of three purifiedS. mutansHKs, i.e., VicK, CiaH, and LiaS. AlthoughS. mutansdoes not have any essential HK but only an essential response regulator, VicR, WKM C showed an MIC of 6.25 μg/ml. This inhibitory effect of WKM C suggests that blocking the autophosphorylation of multiple HKs may inhibit phosphotransfer to VicR from VicK and other HKs. When WKM C was added at sub-MIC levels, the cells formed abnormal biofilms and also showed a defect in competence. When the cells were pretreated with WKM C, an increase in acid sensitivity was observed. Our results show that WKM C represses two pathogenic phenotypes ofS. mutans, indicating the possibility of developing histidine kinase inhibitors into antivirulence drugs.

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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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Antifungal Properties and Target Evaluation of Three Putative Bacterial Histidine Kinase Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.7.1700 ◽

1999 ◽

Vol 43 (7) ◽

pp. 1700-1703 ◽

Cited By ~ 22

Author(s):

Robert J. Deschenes ◽

Hong Lin ◽

Addison D. Ault ◽

Jan S. Fassler

Keyword(s):

Histidine Kinase ◽

Drug Targets ◽

Kinase Inhibitors ◽

Inhibitory Concentration ◽

Drug Efficacy ◽

Antibacterial Drug ◽

Histidine Kinases ◽

Antifungal Properties ◽

Two Component

ABSTRACT Histidine protein kinases have been explored as potential antibacterial drug targets. The recent identification of two-component histidine kinases in fungi has led us to investigate the antifungal properties of three bacterial histidine kinase inhibitors (RWJ-49815, RWJ-49968, and RWJ-61907). All three compounds were found to inhibit growth of the Saccharomyces cerevisiae and Candida albicans strains, with MICs ranging from 1 to 20 μg/ml. However, deletion of SLN1, the only histidine kinase inS. cerevisiae, did not alter drug efficacy. In vitro kinase assays were performed by using the Sln1 histidine kinase purified from bacteria as a fusion protein to glutathione S-transferase. RWJ-49815 and RWJ-49968 inhibited kinase a 50% inhibitory concentration of 10 μM, whereas RWJ-61907 failed to inhibit at concentrations up to 100 μM. Based on these results, we conclude that these compounds have antifungal properties; however, their mode of action appears to be independent of histidine kinase inhibition.

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KinaFrag explores the kinase-ligand fragment interaction space for selective kinase inhibitor discovery

10.1101/2021.03.28.436965 ◽

2021 ◽

Author(s):

Zhi-Zheng Wang ◽

Xing-Xing Shi ◽

Fan Wang ◽

Ge-Fei Hao ◽

Guang-Fu Yang

Keyword(s):

Drug Targets ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Structural Information ◽

3D Structure ◽

Interaction Space ◽

Design Strategies ◽

Growing Strategy ◽

Fragment Interaction

Protein kinases play a crucial role in many cellular signaling processes, making them one of the most important families of drug targets. But selectivity put a barrier at the design of kinase inhibitors. Fragment-based drug design strategies have been successfully applied to develop novel selective kinase inhibitors. However, the complicate kinase-fragment interaction and fragment-to-lead process pose challenges to fragment-based kinase discovery. Here, we developed a web source KinaFrag to investigate kinase-fragment interaction space and perform fragment-to-lead optimization. KinaFrag contained 31464 fragments from reported kinase inhibitors, which involved 3244 crystal fragment structures and 7783 crystal kinase-fragment complexes. These crystal fragments were classified by their binding cleft and subpockets, and their 3D structure and interactions were displayed in KinaFrag. In addition, the structural information, physicochemical information, similarity information, and substructure relationship information were contained in KinaFrag. Moreover, a computational fragment growing strategy obviously developed by our group was implemented in the KinaFrag. We test this fragment growing strategy using our fragment libraries, and obtained a lead compound of c-Met with ~1000-fold in vitro activity improvement compared with the hit compound. We hope KinaFrag could become a powerful tool for the fragment-based kinase inhibitor design. KinaFrag is freely available at http://chemyang.ccnu.edu.cn/ccb/database/KinaFrag/.

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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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Glycation of Host Proteins Increases Pathogenic Potential of Porphyromonas gingivalis

International Journal of Molecular Sciences ◽

10.3390/ijms222112084 ◽

2021 ◽

Vol 22 (21) ◽

pp. 12084

Author(s):

Michał Śmiga ◽

John W. Smalley ◽

Paulina Ślęzak ◽

Jason L. Brown ◽

Klaudia Siemińska ◽

...

Keyword(s):

Porphyromonas Gingivalis ◽

Biofilm Formation ◽

Disease Process ◽

Human Keratinocytes ◽

Bacterial Biofilm ◽

Pathogenic Potential ◽

Glycated Proteins ◽

Sds Page

The non-enzymatic addition of glucose (glycation) to circulatory and tissue proteins is a ubiquitous pathophysiological consequence of hyperglycemia in diabetes. Given the high incidence of periodontitis and diabetes and the emerging link between these conditions, it is of crucial importance to define the basic virulence mechanisms employed by periodontopathogens such as Porphyromonas gingivalis in mediating the disease process. The aim of this study was to determine whether glycated proteins are more easily utilized by P. gingivalis to stimulate growth and promote the pathogenic potential of this bacterium. We analyzed the properties of three commonly encountered proteins in the periodontal environment that are known to become glycated and that may serve as either protein substrates or easily accessible heme sources. In vitro glycated proteins were characterized using colorimetric assays, mass spectrometry, far- and near-UV circular dichroism and UV–visible spectroscopic analyses and SDS-PAGE. The interaction of glycated hemoglobin, serum albumin and type one collagen with P. gingivalis cells or HmuY protein was examined using spectroscopic methods, SDS-PAGE and co-culturing P. gingivalis with human keratinocytes. We found that glycation increases the ability of P. gingivalis to acquire heme from hemoglobin, mostly due to heme sequestration by the HmuY hemophore-like protein. We also found an increase in biofilm formation on glycated collagen-coated abiotic surfaces. We conclude that glycation might promote the virulence of P. gingivalis by making heme more available from hemoglobin and facilitating bacterial biofilm formation, thus increasing P. gingivalis pathogenic potential in vivo.

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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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Animal model for angiotensin II effects in the internal anal sphincter smooth muscle: mechanism of action

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00207.2001 ◽

2002 ◽

Vol 282 (3) ◽

pp. G461-G469 ◽

Cited By ~ 13

Author(s):

Ya-Ping Fan ◽

Rajinder N. Puri ◽

Satish Rattan

Keyword(s):

Smooth Muscle ◽

Protein Kinase ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Rho Kinase ◽

Ang Ii ◽

Kinase C ◽

Rho Kinase Inhibitor ◽

Isolated Smooth Muscle Cells

Effect of ANG II was investigated in in vitro smooth muscle strips and in isolated smooth muscle cells (SMC). Among different species, rat internal and sphincter (IAS) smooth muscle showed significant and reproducible contraction that remained unmodified by different neurohumoral inhibitors. The AT1antagonist losartan but not AT2 antagonist PD-123319 antagonized ANG II-induced contraction of the IAS smooth muscle and SMC. ANG II-induced contraction of rat IAS smooth muscle and SMC was attenuated by tyrosine kinase inhibitors genistein and tyrphostin, protein kinase C (PKC) inhibitor H-7, Ca2+ channel blocker nicardipine, Rho kinase inhibitor Y-27632 or p44/42mitogen-activating protein kinase (MAPK44/42) inhibitor PD-98059. Combinations of nicardipine and H-7, Y-27632, and PD-98059 caused further attenuation of the ANG II effects. Western blot analyses revealed the presence of both AT1 and AT2receptors. We conclude that ANG II causes contraction of rat IAS smooth muscle by the activation of AT1 receptors at the SMC and involves multiple intracellular pathways, influx of Ca2+, and activation of PKC, Rho kinase, and MAPK44/42.

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TEL/PDGFβR Induces Hematologic Malignancies in Mice That Respond to a Specific Tyrosine Kinase Inhibitor

Blood ◽

10.1182/blood.v93.5.1707 ◽

1999 ◽

Vol 93 (5) ◽

pp. 1707-1714 ◽

Cited By ~ 80

Author(s):

Michael H. Tomasson ◽

Ifor R. Williams ◽

Robert Hasserjian ◽

Chirayu Udomsakdi ◽

Shannon M. McGrath ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitor ◽

Tyrosine Kinases ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Transgenic Animals ◽

Myeloid Lineage ◽

Protein Tyrosine

Abstract The TEL/PDGFβR fusion protein is expressed as the consequence of a recurring t(5;12) translocation associated with chronic myelomonocytic leukemia (CMML). Unlike other activated protein tyrosine kinases associated with hematopoietic malignancies, TEL/PDGFβR is invariably associated with a myeloid leukemia phenotype in humans. To test the transforming properties of TEL/PDGFβR in vivo, and to analyze the basis for myeloid lineage specificity in humans, we constructed transgenic mice with TEL/PDGFβR expression driven by a lymphoid-specific immunoglobulin enhancer-promoter cassette. These mice developed lymphoblastic lymphomas of both T and B lineage, demonstrating that TEL/PDGFβR is a transforming protein in vivo, and that the transforming ability of this fusion is not inherently restricted to the myeloid lineage. Treatment of TEL/PDGFβR transgenic animals with a protein tyrosine kinase inhibitor with in vitro activity against PDGFβR (CGP57148) resulted in suppression of disease and a prolongation of survival. A therapeutic benefit was apparent both in animals treated before the development of overt clonal disease and in animals transplanted with clonal tumor cells. These results suggest that small-molecule tyrosine kinase inhibitors may be effective treatment for activated tyrosine kinase–mediated malignancies both early in the course of disease and after the development of additional transforming mutations.

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