(S)-13-[(Dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16,21-dimetheno- 1H,13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-dione (LY333531) and Related Analogues:  Isozyme Selective Inhibitors of Protein Kinase Cβ

Abstract

Protein kinase C (PKC) is a family of closely related serine and threonine kinases. Overactivation of some PKC isozymes has been postulated to occur in several diseases states, including diabetic complications. Selective inhibition of overactivated PKC isozymes may offer a unique therapeutic approach to disease states such as diabetic retinopathy. A novel series of 14-membered macrocycles containing a N-N'-bridged bisindolylmaleimide moiety is described. A panel of eight cloned human PKC isozymes (alpha, beta I, beta II, gamma, delta, epsilon, sigma, eta) was used to identify the series and optimize the structure and associated activity relationship. The dimethylamine analogue LY333531 (1), (S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16, 21-dimetheno-1H, 13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene++ +-1,3(2H)-dione, inhibits the PKC beta I (IC50 = 4.7 nM) and PKC beta II (IC50 = 5.9 nM) isozymes and was 76- and 61-fold selective for inhibition of PKC beta I and PKC beta II in comparison to PKC alpha, respectively. The additional analogues described in the series are also selective inhibitors of PKC beta. LY333531 (1) exhibits ATP dependent competitive inhibition of PKC beta I and is selective for PKC in comparison to other ATP dependent kinases (protein kinase A, calcium calmodulin, caesin kinase, src tyrosine kinase). The cellular activity of the series was assessed using bovine retinal capillary endothelial cells. Retinal endothelial cell dysfunction has been implicated in the development of diabetic retinopathy. Plasminogen activator activity stimulated by a phorbol ester (4 beta-phorbol 12,13-dibutyrate) in endothelial cells was inhibited by the compounds in the series with ED50 values ranging from 7.5 to 0.21 microM. A comparison of the PKC isozyme and related ATP dependent kinase inhibition profiles is provided for the series and compared to the profile for staurosporine, a nonselective PKC inhibitor. The cellular activity of the series is compared with that of the kinase inhibitor staurosporine.

Deregulation of the Ras-Erk Signaling Axis Modulates the Enhancer Landscape

“…Prkcb is frequently amplified in many malignancies and encodes a kinase that is considered a potential therapeutic target in cancer, diabetes, and heart disease (Mochly-Rosen et al, 2012). We hypothesized that HRas G12V MEFs would be more sensitive to LY333531, a clinically relevant Prkcb inhibitor also known as Ruboxistaurin (Jirousek et al, 1996), than cell lines lacking H3K27ac at the Prkcb locus. Indeed, HRas G12V MEFs were significantly more sensitive to doses of LY333531 ranging from 10-15 μM, compared to Spry124 fl/fl EV or Spry124 −/− EV MEFs (Figures 7A and S7E).…”

A ligand-based approach to investigate the expression and function of angiotensin converting enzyme in intact human umbilical vein endothelial cells

“…The general PKC inhibitor GF109203x (0.5-5 µM) and that of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 13 calcium sensitive isoforms Gö6976 (3 µM) both reduced the effect of PMA on ACE density without affecting the baseline expression of the enzyme in the absence of PMA. LY 379196 is a close analog of GF 109203x that has been developed [15]. It had no effect of PMA-induced ACE regulation in a concentration range that is believed to be isoformspecific.…”

Endothelial Nitric Oxide Synthase Phosphorylation at Threonine 495 and Mitochondrial Reactive Oxygen Species Formation in Response to a High H₂O₂ Concentration

“…Nevertheless, as it is shown in figure 4, neither the PKC-β inhibitor RBX nor the general PKC inhibitor GFX was able to inhibit the H 2 O 2 -induced eNOS Thr 495 phosphorylation seen in HUVEC cells. Although RBX was used in a relatively low concentration (50 n M ) in our experiments compared to other studies, the used concentration is approximately 10 times higher than the IC 50 value first described by Jirousek et al [43] and is therefore supposed to completely inhibit the action of both PKC-βI and PKC-βII [42]. The concentration of the general PKC inhibitor GFX of 1 µ M used in our study was well over the reported IC 50 value of 10 n M [42] and we would therefore have expected an effect if PKC was involved in H 2 O 2 -mediated Thr 495 phosphorylation.…”