J. Perry Hall

Pharmacologic Inhibition of Tpl2 Blocks Inflammatory Responses in Primary Human Monocytes, Synoviocytes, and Blood

Tumor necrosis factor α (TNFα) 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 TNFα, interleukin (IL)-1β, and bacterial endotoxin (lipopolysaccharide (LPS)). Moreover, Tpl2 is required for TNFα 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-1β-induced TNFα 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 E2, 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 TNFα 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.

Selective inhibitors of tumor progression loci-2 (Tpl2) kinase with potent inhibition of TNF-α production in human whole blood

Tpl2 (cot/MAP3K8) is an upstream kinase of MEK in the ERK pathway. It plays an important role in Tumor Necrosis Factor-alpha (TNF-alpha) production and signaling. We have discovered that 8-halo-4-(3-chloro-4-fluoro-phenylamino)-6-[(1H-[1,2,3]triazol-4-ylmethyl)-amino]-quinoline-3-carbonitriles (4) are potent inhibitors of this enzyme. In order to improve the inhibition of TNF-alpha production in LPS-stimulated human blood, a series of analogs with a variety of substitutions around the triazole moiety were studied. We found that a cyclic amine group appended to the triazole ring could considerably enhance potency, aqueous solubility, and cell membrane permeability. Optimization of these cyclic amine groups led to the identification of 8-chloro-4-(3-chloro-4-fluorophenylamino)-6-((1-(1-ethylpiperidin-4-yl)-1H-1,2,3-triazol-4-yl)methylamino)quinoline-3-carbonitrile (34). In a LPS-stimulated rat inflammation model, compound 34 showed good efficacy in inhibiting TNF-alpha production.

Mice lacking Tbk1 activity exhibit immune cell infiltrates in multiple tissues and increased susceptibility to LPS-induced lethality

TBK1 is critical for immunity against microbial pathogens that activate TLR4‐ and TLR3‐dependent signaling pathways. To address the role of TBK1 in inflammation, mice were generated that harbor two copies of a mutant Tbk1 allele. This Tbk1Δ allele encodes a truncated Tbk1Δ protein that is catalytically inactive and expressed at very low levels. Upon LPS stimulation, macrophages from Tbk1Δ/Δ mice produce normal levels of proinflammatory cytokines (e.g., TNF‐α), but IFN‐β and RANTES expression and IRF3 DNA‐binding activity are ablated. Three‐month‐old Tbk1Δ/Δ mice exhibit mononuclear and granulomatous cell infiltrates in multiple organs and inflammatory cell infiltrates in their skin, and they harbor a 2‐fold greater amount of circulating monocytes than their Tbk1+/+ and Tbk1+/Δ littermates. Skin from 2‐week‐old Tbk1Δ/Δ mice is characterized by reactive changes, including hyperkeratosis, hyperplasia, necrosis, inflammatory cell infiltrates, and edema. In response to LPS challenge, 3‐month‐old Tbk1Δ/Δ mice die more quickly and in greater numbers than their Tbk1+/+ and Tbk1+/Δ counterparts. This lethality is accompanied by an overproduction of several proinflammatory cytokines in the serum of Tbk1Δ/Δ mice, including TNF‐α, GM‐CSF, IL‐6, and KC. This overproduction of serum cytokines in Tbk1Δ/Δ mice following LPS challenge and their increased susceptibility to LPS‐induced lethality may result from the reactions of their larger circulating monocyte compartment and their greater numbers of extravasated immune cells.

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.

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.

Stimulation of TLR4 by recombinant HSP70 requires structural integrity of the HSP70 protein itself

BackgroundToll-like receptor 4 (TLR4) is activated by bacterial endotoxin, a pathogen-associated molecular pattern (PAMP). It has been suggested that TLR4 can also be activated by damage-associated molecular pattern (DAMP) proteins such as HSP70. It remains a challenge to provide unequivocal evidence that DAMP proteins themselves play a role in TLR4 activation, as the DAMP proteins used are often contaminated with endotoxin and other TLR ligands introduced during protein expression and/or purification.ResultsHere we report that the activation of TLR4 on primary human macrophage cultures by recombinant HSP70 is not solely due to contaminating endotoxin. Polymyxin B pretreatment of HSP70 preparations to neutralize contaminating endotoxin caused significant reductions in the amount of TNF-α induced by the recombinant protein. However, digestion of HSP70 with Proteinase K-agarose beads also dramatically reduced the TNF-α response of macrophages to HSP70, while leaving levels of contaminating endotoxin largely unchanged relative to controls.ConclusionsThese results indicate that the stimulatory effect of recombinant HSP70 requires both the presence of endotoxin and structural integrity of the heat shock protein itself.

TRIL Is Involved in Cytokine Production in the Brain following Escherichia coli Infection

TLR4 interactor with leucine-rich repeats (TRIL) is a brain-enriched accessory protein that is important in TLR3 and TLR4 signaling. In this study, we generated Tril−/− mice and examined TLR responses in vitro and in vivo. We found a role for TRIL in both TLR4 and TLR3 signaling in mixed glial cells, consistent with the high level of expression of TRIL in these cells. We also found that TRIL is a modulator of the innate immune response to LPS challenge and Escherichia coli infection in vivo. Tril−/− mice produce lower levels of multiple proinflammatory cytokines and chemokines specifically within the brain after E. coli and LPS challenge. Collectively, these data uncover TRIL as a mediator of innate immune responses within the brain, where it enhances neuronal cytokine responses to infection.