In vitro activity: Ro 31-8220 inhibits rat brain PKC activity with IC50 of 23 nM, and does not show any high degree of selectivity between PKC-α, PKC-β, PKC-γ, and PKC-ε. Ro 31-8220 also inhibits MSK1, MAPKAPK1, RSK, GSK3β and S6K1 with a potency similar to that for PKC. In addition, Ro 31-8220 inhibits voltage-dependent Na+ channels. Ro 31-8220 alters cellular protein kinase C localization and potently inhibits growth of A549 and MCF-7 cells with IC50 of 0.78 μM and 0.897 μM, respectively. RO 31-8220 enhances epinephrine-induced platelet aggregation in catecholamine hypo-responsive platelets by enhancing Akt phosphorylation. Ro 31-8220 significantly decreases apoE secretion from primary human macrophages by inhibiting vesicular transport of apoE to the plasma membrane without significantly affecting apoE mRNA or apoE protein levels.

Kinase Assay: Assay mixtures contain 0.2 mg/mL peptide-gamma, 10 μM MgCl2, 0.6 mM CaCl2, 10 μM [γ-32P]ATP, 1.25 mg/mL phosphatidylserine and 1.25 ng/mL phorbol 12-myristate 13-acetate in 20 mM Hepes (pH 7.5), 2 mM EDTA, 1 mM dithiothreitol and 0.02% (w/v) Triton X-100. Peptide-γ is a synthetic peptide, GPRPLFCRKGSLRQKW, resembling the PKC-γ pseudosubstrate site, except that a serine residue replaces the pseudosubstrate alanine, converting the peptide from an inhibitor into a substrat. The assays are started by the addition of 2.5 m-units of enzyme, incubated at 30 °C for 10 min and terminated by spotting on to P81 paper, followed by extensive washing in 75 mM orthophosphoric acid. The papers are then washed in ethanol, dried, and incorporated radioactivity is determined by liquidscintillation spectroscopy.

Cell Assay: Human A549 lung and MCF-7 breast carcinoma cells are obtained from the European Collection of Animal Cell Cultures. Cells (passage number 10-30) are cultured in an atmosphere of 5% carbon dioxide, the former in Ham's F-12 medium with penicillin/streptomycin, the latter in minimum essential medium (Eagle's modification) with additional pyruvate (1 mM) and non-essential amino acids. Both media are supplemented with 10% FCS and glutamine (2 mM). Cells are subcultured routinely twice weekly to maintain logarithmic growth. For cell proliferation studies cells are seeded and incubated with 3 ml of medium including agents, which is replenished at intervals of 48 h (A549) or 72 h (MCF-7). Following incubation for 4 days (A549) or 6 days (MCF-7) with drugs, cell number is assessed using a Coulter Counter Model ZM. In order to achieve PKC depletion, cells are incubated for 24 h with bryostatin 1 (1 μM). Under these conditions growth inhibition caused by bryostatin 1 is negligible. Bryostatin is removed by extensive washing of the cells followed by a 2 h recovery period. In previous work using the A549 cell line this washing procedure has been shown to eliminate bryostatin-mediated effects. The cells are then incubated for a further 24 h with staurosporine, RO 31-8220, UCN-01 or H-7. In some experiments cells are incubated with inhibitor for 48 rather than 24 h, in this case bryostation was not removed and left in the incubate. After removal of agents inhibition of DNA synthesis is evaluated by measurement of [3H]Tdr incorporation into cell. Radioactivity is counted using a Packard 1500 Tricarb scintillation counter.