Ro 31-8220

Ro 31-8220 is a selective inhibitor of protein kinase C (PKC) with IC50 values of 5, 24, 14, 27, and 24 nM for PKC α, PKC βI, PKC βII, PKC γ and PKC ε, respectively [1].

PKC is a monomeric Ca2+- and phospholipid-dependent Ser/Thr protein kinases and plays an important role in growth factor-activated signaling and malignant transformation.

Ro 31-8220 is a selective inhibitor of PKC. Also, Ro 31-8220 increased growth factor-stimulated expression of c-Jun but inhibited MKP-1 and c-Fos expression. Ro-31-8220 strongly stimulated the stress-activated protein kinase JNK1 in a PKC-independent way [2]. In peripheral blood mononuclear cells, Ro 31-8220 inhibited the production of mitogen-induced interleukin (IL) -2 and IL-2-dependent T lymphoblast proliferation with IC50 values of 80 and 350 nM, respectively. Also, Ro 31-8220 (400 nM) inhibited IL-2Rα (CD25) expression by 27%, which suggested that Ro 31-8220 inhibited early and late T cell activation [3].

In rat adipocytes and L6 myotubes, RO 31-8220 activated c-Jun N-terminal kinase (JNK) and glycogen synthase (GS) and stimulated glucose incorporation into glycogen. While, RO 31-8220 inhibited the activity of extracellular response kinases 1 and 2 (ERK1/2) in a PKC-independent way [4].

References:
[1].  Wilkinson SE, Parker PJ, Nixon JS. Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C. Biochem J, 1993, 294 ( Pt 2): 335-337.
[2].  Beltman J, McCormick F, Cook SJ. The selective protein kinase C inhibitor, Ro-31-8220, inhibits mitogen-activated protein kinase phosphatase-1 (MKP-1) expression, induces c-Jun expression, and activates Jun N-terminal kinase. J Biol Chem, 1996, 271(43): 27018-27024.
[3].  Geiselhart L, Conti DJ, Freed BM. RO 31-8220, a novel protein kinase C inhibitor, inhibits early and late T cell activation events. Transplantation, 1996, 61(11): 1637-1642.
[4].  Standaert ML, Bandyopadhyay G, Antwi EK, et al. RO 31-8220 activates c-Jun N-terminal kinase and glycogen synthase in rat adipocytes and L6 myotubes. Comparison to actions of insulin.

PKC inhibitors RO 31-8220 and Go 6983 enhance epinephrine-induced platelet aggregation in catecholamine hypo-responsive platelets by enhancing Akt phosphorylation.[Pubmed:21345315]

BMB Rep. 2011 Feb;44(2):140-5.

Impaired responsiveness of platelets to epinephrine (epi) and other catecholamines (CA) has been reported in approximately 20% of the healthy Korean and Japanese populations. In the present study, platelet aggregation induced by epi was potentiated by Ro 31-8220 (RO) or Go 6983 (Go). Phosphorylated Akt (p-Akt) was very low in epi-stimulated PRP from CA-hypo-responders (CA-HY), whereas it was detected in those from CA-good responders (CA-GR). RO and Go increased p-Akt, one of the major downstream effectors of phosphoinositol-3 kinase (PI3K), in epi-stimulated PRP from both groups. Wortmannin, a PI3K inhibitor, attenuated the RO or Go-induced potentiation of p-Akt in epi-stimulated PRP, suggesting positive effects for RO and Go on PI3K. TXA(2) formation was increased by the addition of either RO or Go in epi-stimulated platelets. The present data also suggest that impaired Akt phosphorylation may be responsible for epinephrine hypo-responsiveness of platelets.

Ro 31-6045, the inactive analogue of the protein kinase C inhibitor Ro 31-8220, blocks in vivo activation of p70(s6k)/p85(s6k): implications for the analysis of S6K signalling.[Pubmed:12023032]

FEBS Lett. 2002 May 22;519(1-3):135-40.

The mitogen-stimulated protein kinase p70(s6k)/p85(s6k) (S6K) plays an essential role in cell proliferation and growth, with inhibitors of the S6K signalling pathway showing promise as anti-tumour therapeutics. Here, we report that the bisindolylmaleimide derivative Ro 31-6045, previously reported to be inactive as a kinase inhibitor, inhibited S6K activity in vivo with an IC50=8 microM. Structure/function analysis using mutant forms of S6K indicates that Ro 31-6045 inhibition is independent of the upstream activator mTOR. Ro 31-6045 will prove useful in elucidating the complex activation mechanism of S6K and its independence from mTOR will allow confirmation of functional data obtained using the mTOR inhibitor rapamycin.

Exendin-4 as a stimulator of rat insulin I gene promoter activity via bZIP/CRE interactions sensitive to serine/threonine protein kinase inhibitor Ro 31-8220.[Pubmed:12021195]

Endocrinology. 2002 Jun;143(6):2303-13.

Signal transduction properties of exendin-4 (Ex-4) underlying its ability to stimulate rat insulin I gene promoter (RIP1) activity were assessed in the pancreatic beta-cell line INS-1. Ex-4 acted via glucagon-like peptide-1 receptors to stimulate RIP1 in a glucose-dependent manner, as measured in cells transfected with a -410-bp RIP1-luciferase construct (RIP1-Luc). The action of Ex-4 was independent of cAMP and PKA because it was not blocked by cotransfection with dominant-negative G alpha(s), was unaffected by pretreatment with the membrane-permeant cAMP antagonist 8-Br-Rp-cAMPS, and remained apparent after treatment with PKA inhibitors H-89 or KT 5720. Similarly, cotransfection with a dominant-negative isoform of the type-2 cAMP-regulated guanine nucleotide exchange factor (Epac2) failed to alter the response to Ex-4. Ro 31-8220, a serine/threonine protein kinase inhibitor that targets PKC as as well as the 90-kDa ribosomal S6 kinase (RSK) and mitogen- and stress-activated protein kinase (MSK) family of cAMP response element-binding protein (CREB) kinases, blocked the stimulatory action of Ex-4 at RIP1-Luc. However, selective inhibition of PKC using K-252c, prolonged exposure to phorbol 1,2-myristate-13-acetate, or cotransfection with dominant-negative atypical PKC-zeta, was without effect. A-CREB, a dominant-negative inhibitor of basic region-leucine zipper transcription factors (bZIPs) related in structure to CREB, inhibited the action of Ex-4 at RIP1-Luc, whereas A-ATF-2 was ineffective. Similarly, introduction of deletions at the RIP1 cAMP response element (CRE), or truncation of RIP1 to remove the CRE, nearly abolished the action of Ex-4. Inactivating mutations introduced at the A4/A3 elements, binding sites for the glucose-regulated homeodomain transcription factor PDX-1, did not diminish the response to Ex-4, although a marked reduction of basal promoter activity was observed. The glucose-dependent stimulation of RIP1-Luc by Ex-4 was reproduced using a synthetic reporter (RIP1-CRE-Luc) incorporating multimerized CREs of the RIP1 nonpalindromic sequence 5'-TGACGTCC-3'. It is concluded that the bZIP and CRE-mediated stimulation of RIP1 by Ex-4 explains, at least in part, how this insulinotropic hormone facilitates transcriptional activity of the rat insulin I gene.

Protein Kinase C-Independent Inhibition of Organic Cation Transporter 1 Activity by the Bisindolylmaleimide Ro 31-8220.[Pubmed:26657401]

PLoS One. 2015 Dec 10;10(12):e0144667.

Ro 31-8220 is a potent protein kinase C (PKC) inhibitor belonging to the chemical class of bisindolylmaleimides (BIMs). Various PKC-independent effects of Ro 31-8220 have however been demonstrated, including inhibition of the ATP-binding cassette drug transporter breast cancer resistance protein. In the present study, we reported that the BIM also blocks activity of the solute carrier organic cation transporter (OCT) 1, involved in uptake of marketed drugs in the liver, in a PKC-independent manner. Ro 31-8220, in contrast to other pan-PKC inhibitors such as staurosporine and chelerythrine, was thus shown to cis-inhibit uptake of the reference OCT1 substrate tetraethylammonium in OCT1-transfected HEK293 cells in a concentration-dependent manner (IC50 = 0.18 muM) and without altering membrane expression of OCT1. This blockage of OCT1 was also observed in human hepatic HepaRG cells that constitutionally express OCT1. It likely occurred through a mixed mechanism of inhibition. Ro 31-8220 additionally trans-inhibited TEA uptake in OCT1-transfected HEK293 cells, which likely discards a transport of Ro 31-8220 by OCT1. Besides Ro 31-8220, 7 additional BIMs, including the PKC inhibitor LY 333531, inhibited OCT1 activity, whereas 4 other BIMs were without effect. In silico analysis of structure-activity relationships next revealed that various molecular descriptors, especially 3D-WHIM descriptors related to total size, correspond to key physico-chemical parameters for inhibition of OCT1 activity by BIMs. In addition to activity of OCT1, Ro 31-8220 inhibited those of other organic cation transporters such as multidrug and toxin extrusion protein (MATE) 1 and MATE2-K, whereas, by contrast, it stimulated that of OCT2. Taken together, these data extend the nature of cellular off-targets of the BIM Ro 31-8220 to OCT1 and other organic cation transporters, which has likely to be kept in mind when using Ro 31-8220 and other BIMs as PKC inhibitors in experimental or clinical studies.

Ro 31-8220 is a potent PKC inhibitor, with IC50s of 5, 24, 14, 27, 24 and 23 nM for PKCα, PKCβI, PKCβII, PKCγ, PKCε and rat brain PKC, respectively. Ro 31-8220 also significantly inhibits MAPKAP-K1b, MSK1, S6K1 and GSK3β (IC50s, 3, 8, 15, and 38 nM, respectively), with no effect on MKK3, MKK4, MKK6 and MKK7.

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