Molecular Basis for p38 Protein Kinase Inhibitor Specificity

Lisnock, JeanMarie; Tebben, Andy; Frantz, Betsy; O’Neill, Edward A.; Croft, Gist F.; O’Keefe, Stephen J.; Li, Bing; Hacker, Candice; Laszlo, Stephen de; Smith, Anthony J.; Libby, Brian; Liverton, Nigel J.; Hermes, Jeffrey D.; LoGrasso, Philip V.

doi:10.1021/bi995068+

Cited by 6 publications

(8 citation statements)

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“…A key threonine residue in this pocket (T106 of human p38 kinase) has been shown to interact with halophenyl substituents from this structural class of compounds. Substitution of this threonine residue with amino acids carrying bulky side chains such as glutamine or methionine (T106Q, T106M) prevented inhibitor binding and resulted in an enzyme that was insensitive to this class of compounds (12,22). Our modeling of parasite PKGs implicated the analogous residues T770 of Et-PKG and T761 of Tg-PKG as potentially crucial for the stabilization of the fluorophenyl moiety of compound 1 (Fig.…”

Section: Resultsmentioning

confidence: 94%

“…Primary amino acid sequence alignments of assorted PKG proteins reveal that animal homologues contain a methionine in the positions corresponding to Tg-PKG T761 or Et-PKG T770 (13). From the mutagenesis studies described here for PKG and those published for p38 (12,22), we know that a bulky side chain in this position will interfere with binding of the compound. Indeed, PKG partially purified from chicken displays at least a 1,000-fold lower affinity for compound 1 than parasite PKG (13).…”

Section: Vol 1 2002 Coccidian Parasite Kinase As a Therapeutic Targmentioning

confidence: 83%

“…Guided by the results of previous investigations of the p38 MAP kinase (12,22), an enzyme that is inhibited by trisubstituted imidazoles that are structurally related to compound 1, we targeted a conserved catalytic-site threonine in PKG from both T. gondii and E. tenella. Our predictions were fulfilled by the observations that amino acid residues T761 of Tg-PKG and T770 of Et-PKG are critical for the binding of compound 1 (Table 1).…”

Section: Discussionmentioning

confidence: 99%

“…Animal PKG and p38 kinases are the most obvious candidates as enzymes most likely to exhibit off-target interactions with compound 1. PKG is the most closely related host enzyme based on amino acid sequence similarity (13), and p38 enzymes are inhibited by compounds structurally related to compound 1 (12,22). Primary amino acid sequence alignments of assorted PKG proteins reveal that animal homologues contain a methionine in the positions corresponding to Tg-PKG T761 or Et-PKG T770 (13).…”

Section: Vol 1 2002 Coccidian Parasite Kinase As a Therapeutic Targmentioning

confidence: 99%

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Toxoplasma gondii Cyclic GMP-Dependent Kinase: Chemotherapeutic Targeting of an Essential Parasite Protein Kinase

Donald¹,

Allocco²,

Singh³

et al. 2002

Eukaryot Cell

137

128

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The trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine (compound 1) has in vivo activity against the apicomplexan parasites Toxoplasma gondii and Eimeria tenella in animal models. The presumptive molecular target of this compound in E. tenella is cyclic GMP-dependent protein kinase (PKG). Native PKG purified from T. gondii has kinetic and pharmacologic properties similar to those of the E. tenella homologue, and both have been functionally expressed as recombinant proteins in T. gondii. Computer modeling of parasite PKG was used to predict catalytic site amino acid residues that interact with compound 1. The recombinant laboratory-generated mutants T. gondii PKG T761Q or T761M and the analogous E. tenella T770 alleles have reduced binding affinity for, and are not inhibited by, compound 1. By all other criteria, PKG with this class of catalytic site substitution is indistinguishable from wild-type enzyme. A genetic disruption of T. gondii PKG can only be achieved if a complementing copy of PKG is provided in trans, arguing that PKG is an essential protein. Strains of T. gondii, disrupted at the genomic PKG locus and dependent upon the T. gondii T761-substituted PKGs, are as virulent as wild type in mice. However, unlike mice infected with wild-type T. gondii that are cured by compound 1, mice infected with the laboratory-generated strains of T. gondii do not respond to treatment. We conclude that PKG represents the primary molecular target responsible for the antiparasitic efficacy of compound 1.

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Section: Resultsmentioning

confidence: 94%

Section: Vol 1 2002 Coccidian Parasite Kinase As a Therapeutic Targmentioning

confidence: 83%

Section: Discussionmentioning

confidence: 99%

Section: Vol 1 2002 Coccidian Parasite Kinase As a Therapeutic Targmentioning

confidence: 99%

See 2 more Smart Citations

Toxoplasma gondii Cyclic GMP-Dependent Kinase: Chemotherapeutic Targeting of an Essential Parasite Protein Kinase

Donald¹,

Allocco²,

Singh³

et al. 2002

Eukaryot Cell

137

128

View full text Add to dashboard Cite

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“…However, this analysis confirmed the reporter data indicating that neither the p38 nor the ERK MAPK pathways play a major role in NF-jB-dependent transcription in these cells. Finally, it should be noted that the use of the p38 MAPK inhibitor, SB203580, only implicates the a and b isofroms of p38, as SB203580 has little or no effect on p38 c and d and it is possible that this could explain discrepancies between over-expression and inhibitor based studies [34].…”

Section: Discussionmentioning

confidence: 99%

ICAM‐1 expression is highly NF‐κB‐dependent in A549 cells

Holden

Catley

Cambridge

et al. 2004

European Journal of Biochemistry

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The transcription factor nuclear factor jB (NF-jB) is an activator of multiple cytokines, chemokines and adhesion molecules, which are important in inflammatory diseases such as asthma, and is consequently considered as an attractive therapeutic target. In the present study, a constitutively active dominant version of IjBa, IjBaDN, was introduced into A549 pulmonary cells by adenovirusmediated delivery. The dominant IjB, but not a null viral vector, prevented the induction of NF-jB-dependent transcription by both tumor necrosis factor a (TNFa) and interleukin-1b (IL-1b). Similarly, both TNFa and IL-1b strongly induced mRNA and protein expression of intercellular adhesion molecule (ICAM)-1 and in each case this was prevented by adenovirus expressing the dominant IjB, but not by the null virus, thereby establishing ICAM-1 as an NF-jB-dependent gene. Numerous studies have suggested key roles for the p38 and extracellular regulated kinase (ERK) mitogen-activated protein kinase (MAPK) cascades in the activation and transactivation of NF-jB. We show here that SB203580, a selective inhibitor of the p38 MAPK, and PD098059 and UO126, both selective inhibitors of the ERK MAPK cascade, have no effect on TNFa or IL-1b-induced translocation and DNA binding of NF-jB. Furthermore, these inhibitors showed no pharmacologically relevant effect on NF-jB-dependent transcription nor was there any effect on expression of ICAM-1. Taken together these data highlight the potential use of inhibition of the NF-jB signalling pathway in pulmonary inflammatory diseases and suggest that inhibitors of the p38 and ERK MAPK pathways may be of lesser effect.

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Small molecular anti-cytokine agents

2005

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The recent successful introduction of the anti-cytokine biologicals Etanercept, Infliximab, Adalimumab, and Anakinra has stimulated the search for anti-cytokine small-molecules. A number of molecular targets have been identified for the development of such small molecular anti-cytokine agents. The focus of this review will be on those inhibitors of cytokine production, which target either p38 mitogen activated protein (MAP) kinase, TNF-alpha converting enzyme (TACE), or IL-1beta converting enzyme (ICE). P38 MAP kinase occupies a central role in the signaling network responsible for the upregulation of proinflammatory cytokines like interleukin 1beta (IL-1beta) and TNF-alpha, and regulates their biosynthesis at both the transcriptional and translational level. TACE and ICE are two proteases required for the processing of proTNF-alpha and proIL-1beta, respectively into their mature, proinflammatory form. Since the mid-1990s, a plethora of inhibitors of p38 MAP kinase, TACE, and ICE has been characterized in vitro, and individual representatives from all three inhibitor classes have in the meantime been advanced into clinical trials. This review will highlight the correlation between effective inhibition at the molecular target and cellular activity in functional assays of cytokine, particularly TNF-alpha and IL-1beta, production. Structure-activity relationships (SAR) will be discussed regarding activity at the respective enzyme target, but also with regard to properties required for efficient in vitro and in vivo cellular activity (e.g., oral availability, solubility, cell penetration, etc.).

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Molecular Basis for p38 Protein Kinase Inhibitor Specificity

Cited by 6 publications

References 0 publications

Toxoplasma gondii Cyclic GMP-Dependent Kinase: Chemotherapeutic Targeting of an Essential Parasite Protein Kinase

Toxoplasma gondii Cyclic GMP-Dependent Kinase: Chemotherapeutic Targeting of an Essential Parasite Protein Kinase

ICAM‐1 expression is highly NF‐κB‐dependent in A549 cells

Small molecular anti-cytokine agents

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