Pharmacophore generation and atom-based 3D-QSAR of novel quinoline-3-carbonitrile derivatives as Tpl2 kinase inhibitors

Tumour progression locus-2 (Tpl2) is a serine/threonine kinase, which regulates the expression of tumour necrosis factor α. The article describes the development of a robust pharmacophore model and the investigation of structure-activity relationship analysis of quinoline-3-carbonitrile derivatives reported for Tpl2 kinase inhibition. A five point pharmacophore model (ADRRR) was developed and used to derive a predictive atom-based 3-dimensional quantitative structure activity relationship (3D-QSAR) model. The obtained 3D-QSAR model has an excellent correlation coefficient value (r(2)= 0.96), Fisher ratio (F = 131.9) and exhibited good predictive power (q(2) = 0.79). The QSAR model suggests that the inclusion of hydrophobic substituents will enhance the Tpl2 kinase inhibition. In addition, H-bond donating groups, negative ionic groups and electron withdrawing groups positively contribute to the Tpl2 kinase inhibition. Further, pharmacophoric model was validated by the receiver operating characteristic curve analysis and was employed for virtual screening to identify six potential Tpl2 kinase inhibitors. The findings of this study provide a set of guidelines for designing compounds with better Tpl2 kinase inhibitory potency.     

Deregulated activation of oncoprotein kinase Tpl2/Cot in HTLV-I-transformed T cells

Protein kinase Tpl2/Cot is encoded by a protooncogene that is cis-activated by retroviral insertion in murine T cell lymphomas. It has remained unclear whether this oncoprotein kinase is mutated or post-translationally activated in human cancer cells. We have shown here that Tpl2/Cot is constitutively activated in human leukemia cell lines transformed by the human T cell leukemia virus type I (HTLV-I). The kinase activity of Tpl2/Cot is normally suppressed through its physical interaction with an inhibitor, the NF-kappaB1 precursor protein p105. Interestingly, a large pool of Tpl2/Cot is liberated from p105 and exhibits constitutive kinase activity in HTLV-I-transformed T cells. In contrast to its labile property in normal cells, the pathologically activated Tpl2/Cot is remarkably stable. Further, whereas the physiological activation of Tpl2/Cot involves its long isoform, the HTLV-activated Tpl2/Cot is predominantly the short isoform. We have also shown that the HTLV-I-encoded Tax protein is able to activate Tpl2/Cot in transfected cells. Finally, Tpl2/Cot participates in the activation of NF-kappaB by Tax. These findings indicate that deregulated activation of Tpl2/Cot may occur in human cancer cells.     

Pharmacophore generation and atom-based 3D-QSAR of novel quinoline-3-carbonitrile derivatives as Tpl2 kinase inhibitors

Tumour progression locus-2 (Tpl2) is a serine/threonine kinase, which regulates the expression of tumour necrosis factor α. The article describes the development of a robust pharmacophore model and the investigation of structure-activity relationship analysis of quinoline-3-carbonitrile derivatives reported for Tpl2 kinase inhibition. A five point pharmacophore model (ADRRR) was developed and used to derive a predictive atom-based 3-dimensional quantitative structure activity relationship (3D-QSAR) model. The obtained 3D-QSAR model has an excellent correlation coefficient value (r²?=?0.96), Fisher ratio (F?=?131.9) and exhibited good predictive power (q²?=?0.79). The QSAR model suggests that the inclusion of hydrophobic substituents will enhance the Tpl2 kinase inhibition. In addition, H-bond donating groups, negative ionic groups and electron withdrawing groups positively contribute to the Tpl2 kinase inhibition. Further, pharmacophoric model was validated by the receiver operating characteristic curve analysis and was employed for virtual screening to identify six potential Tpl2 kinase inhibitors. The findings of this study provide a set of guidelines for designing compounds with better Tpl2 kinase inhibitory potency.     

Pharmacologic inhibition of tpl2 blocks inflammatory responses in primary human monocytes, synoviocytes, and blood

Tumor necrosis factor alpha (TNFalpha) is a pro-inflammatory cytokine that controls the initiation and progression of inflammatory diseases such as rheumatoid arthritis. Tpl2 is a MAPKKK in the MAPK (i.e. ERK) pathway, and the Tpl2-MEK-ERK signaling pathway is activated by the pro-inflammatory mediators TNFalpha, interleukin (IL)-1beta, and bacterial endotoxin (lipopolysaccharide (LPS)). Moreover, Tpl2 is required for TNFalpha expression. Thus, pharmacologic inhibition of Tpl2 should be a valid approach to therapeutic intervention in the pathogenesis of rheumatoid arthritis and other inflammatory diseases in humans. We have developed a series of highly selective and potent Tpl2 inhibitors, and in the present study we have used these inhibitors to demonstrate that the catalytic activity of Tpl2 is required for the LPS-induced activation of MEK and ERK in primary human monocytes. These inhibitors selectively target Tpl2 in these cells, and they block LPS- and IL-1beta-induced TNFalpha production in both primary human monocytes and human blood. In rheumatoid arthritis fibroblast-like synoviocytes these inhibitors block ERK activation, cyclooxygenase-2 expression, and the production of IL-6, IL-8, and prostaglandin E(2), and the matrix metalloproteinases MMP-1 and MMP-3. Taken together, our results show that inhibition of Tpl2 in primary human cell types can decrease the production of TNFalpha and other pro-inflammatory mediators during inflammatory events, and they further support the notion that Tpl2 is an appropriate therapeutic target for rheumatoid arthritis and other human inflammatory diseases.     

Identification of a novel class of selective Tpl2 kinase inhibitors: 4-Alkylamino-[1,7]naphthyridine-3-carbonitriles

We have previously reported the discovery and initial SAR of the [1,7]naphthyridine-3-carbonitriles and quinoline-3-carbonitriles as Tumor Progression Loci-2 (Tpl2) kinase inhibitors. In this paper, we report new SAR efforts which have led to the identification of 4-alkylamino-[1,7]naphthyridine-3-carbonitriles. These compounds show good in vitro and in vivo activity against Tpl2 and improved pharmacokinetic properties. In addition they are highly selective for Tpl2 kinase over other kinases, for example, EGFR, MEK, MK2, and p38. Lead compound 4-cycloheptylamino-6-[(pyridin-3-ylmethyl)-amino]-[1,7]naphthyridine-3-carbonitrile (30) was efficacious in a rat model of LPS-induced TNF-alpha production.     

Discovery of indazoles as inhibitors of Tpl2 kinase

Synthesis, modeling and structure-activity relationship of indazoles as inhibitors of Tpl2 kinase are described. From a high throughput screening effort, we identified an indazole hit compound 5 that has a single digit micromolar Tpl2 activity. Through SAR modifications at the C3 and C5 positions of the indazole, we discovered compound 31 with good potency in LANCE assay and cell-based p-Erk assay.     

Inhibition of Tpl2 kinase and TNF-alpha production with 1,7-naphthyridine-3-carbonitriles: synthesis and structure-activity relationships

The synthesis and structure-activity studies of a series of 6-substituted-4-anilino-[1,7]-naphthyridine-3-carbonitriles as inhibitors of Tpl2 kinase are described. The early exploratory work described here may lead to the discovery of compounds with significant therapeutic potential for treating rheumatoid arthritis and other inflammatory diseases.     

Identification and SAR of a new series of thieno[3,2-d]pyrimidines as Tpl2 kinase inhibitors

We report here the synthesis and SAR of a new series of thieno[3,2-d]pyrimidines as potent Tpl2 kinase inhibitors. The proposed binding mode suggests the potential flipped binding mode depending on the substitution. Biacore studies show evidence of binding of these molecules to the protein kinase. The kinome inhibition profile of these molecules suggests good selectivity.     

The tyrosine kinase Syk regulates TPL2 activation signals

Tpl2/Cot is a serine/threonine kinase that plays a key physiological role in the regulation of immune responses to pro-inflammatory stimuli, including tumor necrosis factor-alpha (TNF-alpha). TNF-alpha stimulates the JNK, ERK, and p38 mitogen-activated protein kinases and the NF-kappaB pathway by recruiting RIP1 and TRAF2 to the TNF receptor 1. Here we showed that Tpl2 activation by TNF-alpha signals depends on the integrity of the Tpl2-interacting proteins RIP1 and TRAF2, which are required for the engagement of the ERK mitogen-activated protein kinase pathway. However, neither RIP1 nor TRAF2 overexpression was sufficient to activate Tpl2 and ERK. We also showed that Tpl2 activation by TNF-alpha depends on a tyrosine kinase activity that is detected in TNF-alpha-stimulated cells. Based on both genetic and biochemical evidence, we concluded that in a variety of cell types, Syk is the tyrosine kinase that plays an important role in the activation of Tpl2 upstream of ERK. These data therefore dissect the TNF receptor 1 proximal events that regulate Tpl2 and ERK and highlight a role for RIP1, TRAF2, and Syk in this pathway.     

Tpl2 (tumor progression locus 2) phosphorylation at Thr290 is induced by lipopolysaccharide via an Ikappa-B Kinase-beta-dependent pathway and is required for Tpl2 activation by external signals

The serine-threonine protein kinase encoded by the tumor progression locus 2 (Tpl2) proto-oncogene transduces Toll-like receptor and death receptor signals in a variety of cell types. Here we show that Tpl2 undergoes phosphorylation at Thr(290) both in cells overexpressing Tpl2 and in cells stimulated with lipopolysaccharide (LPS) or tumor necrosis factor-alpha and that phosphorylation on this site parallels Tpl2 activation. Reconstitution of Tpl2(-/-) macrophages with wild type Tpl2 or Tpl2 T290D restored ERK activation by LPS, whereas reconstitution of the same cells with Tpl2 T290A did not, suggesting that phosphorylation at Thr(290) is required for the physiological activation of Tpl2 by external signals. Both the wild type Tpl2 and the kinase-inactive mutant Tpl2 K167M undergo Thr(290) phosphorylation, suggesting that Thr(290) may be a site of trans-phosphorylation rather than auto-phosphorylation. Pretreatment of 293 cells and primary macrophages with the Ikappa-B kinase-beta (IKKbeta) inhibitor PS-1145 blocked Tpl2 phosphorylation at Thr(290), suggesting that phosphorylation depends on IKKbeta, an obligatory positive regulator of Tpl2. We conclude that Tpl2 phosphorylation at Thr(290) is induced by LPS, depends on IKKbeta, and is required for the physiological activation of Tpl2 by external signals.     

Tumor Progression Locus 2 Promotes Induction of IFNλ, Interferon Stimulated Genes and Antigen-Specific CD8+ T Cell Responses and Protects against Influenza Virus

Mitogen-activated protein kinase (MAP) cascades are important in antiviral immunity through their regulation of interferon (IFN) production as well as virus replication. Although the serine-threonine MAP kinase tumor progression locus 2 (Tpl2/MAP3K8) has been implicated as a key regulator of Type I (IFNα/β) and Type II (IFNγ) IFNs, remarkably little is known about how Tpl2 might contribute to host defense against viruses. Herein, we investigated the role of Tpl2 in antiviral immune responses against influenza virus. We demonstrate that Tpl2 is an integral component of multiple virus sensing pathways, differentially regulating the induction of IFNα/β and IFNλ in a cell-type specific manner. Although Tpl2 is important in the regulation of both IFNα/β and IFNλ, only IFNλ required Tpl2 for its induction during influenza virus infection both in vitro and in vivo. Further studies revealed an unanticipated function for Tpl2 in transducing Type I IFN signals and promoting expression of interferon-stimulated genes (ISGs). Importantly, Tpl2 signaling in nonhematopoietic cells is necessary to limit early virus replication. In addition to early innate alterations, impaired expansion of virus-specific CD8⁺ T cells accompanied delayed viral clearance in Tpl2^-/- mice at late time points. Consistent with its critical role in facilitating both innate and adaptive antiviral responses, Tpl2 is required for restricting morbidity and mortality associated with influenza virus infection. Collectively, these findings establish an essential role for Tpl2 in antiviral host defense mechanisms.     

Tumor progression locus 2 (Tpl2) deficiency does not protect against obesity-induced metabolic disease

Obesity is associated with a state of chronic low grade inflammation that plays an important role in the development of insulin resistance. Tumor progression locus 2 (Tpl2) is a serine/threonine mitogen activated protein kinase kinase kinase (MAP3K) involved in regulating responses to specific inflammatory stimuli. Here we have used mice lacking Tpl2 to examine its role in obesity-associated insulin resistance. Wild type (wt) and tpl2(-/-) mice accumulated comparable amounts of fat and lean mass when fed either a standard chow diet or two different high fat (HF) diets containing either 42% or 59% of energy content derived from fat. No differences in glucose tolerance were observed between wt and tpl2(-/-) mice on any of these diets. Insulin tolerance was similar on both standard chow and 42% HF diets, but was slightly impaired in tpl2(-/-) mice fed the 59% HFD. While gene expression markers of macrophage recruitment and inflammation were increased in the white adipose tissue of HF fed mice compared with standard chow fed mice, no differences were observed between wt and tpl2(-/-) mice. Finally, a HF diet did not increase Tpl2 expression nor did it activate Extracellular Signal-Regulated Kinase 1/2 (ERK1/2), the MAPK downstream of Tpl2. These findings argue that Tpl2 does not play a non-redundant role in obesity-associated metabolic dysfunction.