Expression and purification of active receptor interacting protein 1 kinase using a baculovirus system

Maki, James S.; Brazell, J. Tres; Teng, Xin; Cuny, Gregory D.; Degterev, Alexei

doi:10.1016/j.pep.2013.03.002

Cited by 4 publications

(2 citation statements)

References 25 publications

(33 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In contrast, ponatinib displayed ~3-fold higher activity in vitro than in cells. We have previously optimized the length of RIPK1’s kinase domain (recombinant RIPK1, a.a. 1–327) to maximize its catalytic activity (Maki and Degterev, 2013; Maki et al, 2013) and, hence, the kinase active Glu-in conformation. The Glu-in conformation creates an additional energy barrier (due to the loss of a highly conserved Glu/Lys ionic bond in the Glu-out conformation) that must be overcome by necrostatins (Fig.…”

Section: Resultsmentioning

confidence: 99%

Structure Guided Design of Potent and Selective Ponatinib-Based Hybrid Inhibitors for RIPK1

et al. 2015

Self Cite

View full text Add to dashboard Cite

Summary RIPK1 and RIPK3, two closely related RIPK family members, have emerged as important regulators of pathologic cell death and inflammation. In the current work, we report that the Bcr-Abl inhibitor and anti-leukemia agent ponatinib is also a first-in-class dual inhibitor of RIPK1 and RIPK3. Ponatinib potently inhibited multiple paradigms of RIPK1- and RIPK3-dependent cell death and inflammatory TNFα gene transcription. We further describe design strategies that utilize the ponatinib scaffold to develop two classes of inhibitors (CS and PN series), each with greatly improved selectivity for RIPK1. In particular, we detail the development of PN10, a highly potent and selective ‘hybrid’ RIPK1 inhibitor, capturing the best properties of two different allosteric RIPK1 inhibitors, ponatinib and necrostatin-1. Finally, we show that RIPK1 inhibitors from both classes are powerful blockers of TNF-induced injury in vivo. Altogether, these findings outline promising candidate molecules and design approaches for targeting RIPK1/3-driven inflammatory pathologies.

show abstract

Section: Resultsmentioning

confidence: 99%

Structure Guided Design of Potent and Selective Ponatinib-Based Hybrid Inhibitors for RIPK1

et al. 2015

Self Cite

View full text Add to dashboard Cite

show abstract

“…Both RIP1 and RIP3 are clients of the HSP90-CDC37 cochaperone complex. Although it has been previously reported that RIP1 can be better expressed and purified when coexpressed with CDC37, and an HSP90 inhibitor caused the down-regulation of the RIP1 protein level (22,25,26), it is now clear that not only are both latent RIP1 and RIP3 associated with the HSP90-CDC37 complex, their activation during necroptosis is critically dependent on the activity of such a chaperone complex. Interventions with either chemical HSP90 inhibitors or CDC37 knockdown blocked RIP1-RIP3 interaction, RIP3 autophosphorylation, and the ensuing necroptosis.…”

Section: Discussionmentioning

confidence: 99%

A cytosolic heat shock protein 90 and cochaperone CDC37 complex is required for RIP3 activation during necroptosis

Cao

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

141

131

View full text Add to dashboard Cite

Receptor-interacting protein kinase 3, RIP3, and a pseudokinase mixed lineage kinase-domain like protein, MLKL, constitute the core components of the necroptosis pathway, which causes programmed necrotic death in mammalian cells. Latent RIP3 in the cytosol is activated by several upstream signals including the related kinase RIP1, which transduces signals from the tumor necrosis factor (TNF) family of cytokines. We report here that RIP3 activation following the induction of necroptosis requires the activity of an HSP90 and CDC37 cochaperone complex. This complex physically associates with RIP3. Chemical inhibitors of HSP90 efficiently block necroptosis by preventing RIP3 activation. Cells with knocked down CDC37 were unable to respond to necroptosis stimuli. Moreover, an HSP90 inhibitor that is currently under clinical development as a cancer therapy was able to prevent systemic inflammatory response syndrome in rats treated with TNF-α. HSP90 and CDC37 cochaperone complex-mediated protein folding is thus an important part of the RIP3 activation process during necroptosis.

show abstract

Evaluation of RIP1K and RIP3K expressions in the malignant and benign breast tumors

et al. 2016

View full text Add to dashboard Cite

Receptor-interacting protein kinase 1 (RIP1K) and RIP3K belong to RIPK family, which regulate cell survival and cell death. In the present investigation, the expression levels of RIP1K and RIP3K were evaluated in the 30 malignant, 15 benign, and 20 normal breast tissues, and their correlation with clinicopathological characteristics was also studied. The expression levels of RIP1K and RIP3K were determined, by western blot analysis. The relative RIP1K expression was significantly higher in the malignant and benign tumors when compared to those of normal tissues (P < 0.0001 and P < 0.001, respectively). However, the expression level of RIP3K was significantly lower in the malignant tumors than those of normal and benign values (P < 0.001 and P < 0.01, respectively). Positive significant correlation was found for RIP1K expression with tumor size (P < 0.001), grades (P < 0.0001), and c-erbB2 (P < 0.001), but negative significant correlation was detected with patient's age (P < 0.001), estrogen receptor (ER) (P < 0.001), progesterone receptor (PR) (P < 0.01), and P53 (P<0.01) status. RIP3K expression was significantly lower in the pre-menopauses (P < 0.01), grade III (P < 0.05), ER-negative (P < 0.05), and c-erbB2-negative malignant tumors, but no correlation was detected with tumor size, PR, and P53 status. No significant correlation was observed for RIP1K and RIP3K expressions with Ki67 and Her2. Based on the present results, it is concluded that reduction of RIP3K expression in the malignant breast tumor might be an important evidence to support the antitumor activity of this enzyme in vivo. However, RIP1K expression was shown to be higher in the malignant breast tumors than those of normal and benign breast tissues, which probably designates as a poor prognostic factor.

show abstract

Expression and purification of active receptor interacting protein 1 kinase using a baculovirus system

Cited by 4 publications

References 25 publications

Structure Guided Design of Potent and Selective Ponatinib-Based Hybrid Inhibitors for RIPK1

Structure Guided Design of Potent and Selective Ponatinib-Based Hybrid Inhibitors for RIPK1

A cytosolic heat shock protein 90 and cochaperone CDC37 complex is required for RIP3 activation during necroptosis

Evaluation of RIP1K and RIP3K expressions in the malignant and benign breast tumors

Contact Info

Product

Resources

About