Masaki Goto

Regulation of Vascular Smooth Muscle Cell Proliferation by Nuclear Factor-κB and Its Inhibitor, I-κB

Proliferation of vascular smooth muscle cells (SMC) is a crucial event in the formation of atherosclerotic tissues and is regulated by nuclear transcriptional factors including nuclear factor-κB (NF-κB). We constructed a reporter gene assay to measure NF-κB-dependent transcriptional activity in SMC. Thrombin receptor-activating peptide (TRAP) and basic fibroblast growth factor (bFGF) stimulated SMC proliferation and rapidly enhanced the NF-κB transcriptional activity in a dose-dependent manner. 4-Cyano-5,5-bis-(methoxyphenyl)4-pentenoic acid (E5510) significantly inhibited SMC proliferation and also suppressed NF-κB transcription stimulated by TRAP and bFGF. In contrast, although tumor necrosis factor (TNF)-α activated NF-κB transcription, E5510 had no effect on TNF-α-induced activation. NF-κB was activated after the stimulation of TRAP, bFGF, and TNF-α in electrophoretic mobility shift assay, and E5510 suppressed the NF-κB activation induced by TRAP and bFGF but not the activation by TNF-α. Western blot analysis of I-κBα and I-κBβ, inhibitors of NF-κB, indicated that I-κBα degradation, rather than I-κBβ degradation, was important in NF-κB activation after the stimulation of TRAP and bFGF. PD98059, an inhibitor of extracellular signal-regulated kinase (ERK) kinase, suppressed NF-κB transcriptional activity and SMC proliferation. The phosphorylation of ERK1/2 was rapidly induced by TRAP and bFGF but not by TNF-α. These results indicate that TRAP and bFGF induced I-κB degradation and NF-κB activation through a distinct pathway from TNF-α and that ERK1/2 may play an important role in NF-κB activation induced by TRAP and bFGF.

E6201 [(3S,4R,5Z,8S,9S,11E)-14-(Ethylamino)-8, 9,16-trihydroxy-3,4-dimethyl-3,4,9,19-tetrahydro-1H-2-benzoxacyclotetradecine-1,7(8H)-dione], a Novel Kinase Inhibitor of Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Kinase (MEK)-1 and MEK Kinase-1: In Vitro Characterization of Its Anti-Inflammatory and Antihyperproliferative Activities

The goal of this study is to identify a novel inhibitor with anti-inflammatory and antiproliferative properties for the treatment of psoriasis. Compound f152A1 [(3S,5Z,8S,11E)-8,9,16-trihydroxy-14-methoxy-3-methyl-3,4,9,10-tetrahydro-1H-benzo[c][1]oxacyclotetradecine1,7(8H)-dione] was identified as the main active metabolite with strong inhibitory activity against tumor necrosis factor-α (TNFα) transcription in a fraction originated from the fermentation broth of the fungus Curvularia verruculosa. Although active in cell-based assays, f152A1 was unstable in plasma and liver microsome preparations, thus limiting its pharmaceutical utilization. To improve the metabolic properties of f152A1, a medicinal chemistry program was undertaken, resulting in the generation of over 400 analogs of f152A1. Eventually, E6201 [(3S,4R,5Z,8S,9S,11E)-14-(ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,19-tetrahydro-1H-2-benzoxacyclotetradecine-1,7(8H)-dione] was identified as a promising analog in this series. In the present study, we characterized the in vitro activities of E6201 and discovered that the compound inhibits lipopolysaccharide-activated TNFα reporter activity in THP-1-33 cells with an IC50 value of 50 nM and selectively inhibits mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-1 and MEK kinase-1 in cell-free biochemical assays. In addition, E6201 showed inhibitory activity in several other cell-based systems: 1) phosphorylation of c-jun N-terminal kinase and p38 MAPKs; 2) nuclear factor-κB and activated protein-1 activation in various cell types; 3) interleukin (IL)-2 production from human lymphocytes; 4) hyperproliferation of human keratinocytes; 5) IL-8 production from human keratinocytes; and 6) proinflammatory cytokine production from human peripheral blood mononuclear cells. Based on the data presented here, E6201 may be beneficial for treatment of inflammatory and hyperproliferative diseases such as psoriasis through its anti-inflammatory activities on immune cells and antihyperproliferative activities on keratinocytes.

Discovery of anti-inflammatory clinical candidate E6201, inspired from resorcylic lactone LL-Z1640-2, III.

Inspired by natural product, LL-Z1640-2, clinical candidate, E6201 (22) was discovered in a medicinal chemistry effort through total synthesis. The modification on C14-position to N-alkyl substitution showed to be potent in vitro and orally active in vivo in anti-inflammatory assays.

Discovery of a potent, metabolically stabilized resorcylic lactone as an anti-inflammatory lead

With bioactivity-guided phenotype screenings, a potent anti-inflammatory compound f152A1 has been isolated, characterized and identified as the known natural product LL-Z1640-2. Metabolic instability precluded its use for the study on animal disease models. Via total synthesis, a potent, metabolically stabilized analog ER-803064 has been created; addition of the (S)-Me group at C4 onto f152A1 has resulted in a dramatic improvement on its metabolic stability, while preserving the anti-inflammatory activities.

E6201, a Novel Kinase Inhibitor of Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Kinase-1 and Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Kinase Kinase-1: In Vivo Effects on Cutaneous Inflammatory Responses by Topical Administration

E6201 [(3S,4R,5Z,8S,9S,11E)-14-(ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,10-tetrahydro-1H-2-benzoxacyclotetradecine-1,7(8H)-dione)] is a novel anti-inflammatory agent that has potent inhibitory effects on the production of proinflammatory cytokines from leukocytes and antiproliferative activity on keratinocytes. To characterize the in vivo pharmacological activity of E6201, topically administered E6201 was evaluated in several different animal models of dermatitis. E6201 formulated as an ointment or cream showed dose-dependent inhibition of croton oil-induced acute edema formation and neutrophil infiltration into mouse skin. In addition, E6201 cream inhibited the 1-fluoro-2,4-dinitrobenzene-induced contact hypersensitivity reaction mediated by T cells in mice. In this model, E6201 cream also suppressed the migration of neutrophils and lymphocytes into the inflammatory site. Pretreatment with E6201 cream attenuated phorbol-12 myristate 13-acetate-induced ornithine decarboxylase activity, a marker of proliferation in epidermis. Furthermore, E6201 ointment showed inhibitory effects on both mezerein-induced and interleukin (IL)-23-induced epidermal hyperplasia. E6201 also suppressed T cell receptor-stimulated IL-17 production from human T cells. These results indicate that topically administered E6201 may be a useful agent for the prevention and treatment of cutaneous inflammatory and hyperproliferative diseases such as psoriasis.

Discovery of an in vitro and in vivo potent resorcylic lactone analog of LL-Z1640-2 as anti-inflammatory lead, II

The potent in vitro lead compound, ER-803064 (2), a MEK1 and MEKK1 inhibitor inspired from natural product LL-Z1640-2 (f152A1), was further optimized to improve in vitro and in vivo potency. The modifications on C14 position led to discovery of the lead compounds 28 and 29, which regained full in vitro potency of f152A1 and showed higher in vivo potency by iv administration.

Inhibitory effects of E-5110 on interleukin-1 generation from human monocytes

Effects of E-5110, a novel non-steroidal antiinflammatory drug, on interleukin-1 (IL-1) generation from human monocytes were studiedin vitro. E-5110 reduced the amounts of extra- and intracellular IL-1 activity induced by lipopolysaccharide (LPS, 1 μg/ml) in a dose-dependent manner (1–10μM). E-5110 also inhibited the IL-1 generation induced by antigen-antibody complexes, opsonized zymosan and silica particles. It was suggested that the inhibition of IL-1 generation by E-5110 was independent of the inhibitory effects on arachidonate cyclooxygenase and/or lipoxygenase because indomethacin, piroxicam, BW755C and AA861 had no effects on IL-1 generation. Hydrocortisone (IC50:0.084 μM), aurothioglucose (11.5 μM) and lobenzarit (75.0 μM), which are clinically effective antirheumatic drugs, also inhibited IL-1 generation, like E-5110 (1.21 μM). It is expected that E-5110 will be superior to classical non-steroidal antiinflammatory drugs in medical treatment of rheumatoid arthritis.

Conformational Analyses and MO Studies of f152A1 and Its Analogues as Potent Protein Kinase Inhibitors

f152A1 was isolated from a fermentation broth of Curvularia verruculosa and characterized as a potent inhibitor of TNFalpha transcription, with anti-inflammatory activity. f152A1 and several analogues displayed inhibitory activity against the MAP kinases ERK2 and MEK1 in in vitro kinase assays. Through SAR studies on f152A1 and analogues prepared via total synthesis, we have identified structural features that contribute to inhibitory activity. To rationalize these results and to aid in the discovery process, a combination of high temperature molecular dynamics and MOPAC AM1 semiempirical molecular orbital method studies was used in studies that yielded a postulated active conformation, M1(8). This active conformation M1(8) reflects a high degree of conformational similarity among f152A1 and its more potent analogues. In view of the highly reactive cis-enone moiety in the flexible 14-membered resorcylic acid lactone ring of f152A1, the chemical reactivities of the enone moieties in various analogues were assessed by molecular orbital calculations. The enone reactivity analyses suggested that these inhibitors were prone to Michael addition at the alpha,beta-unsaturated ketone moiety and might chemically react with cysteine residues in the ATP-binding site of MAP kinases. Reactivity of the cis-enone moiety and the M1(8) conformation make important contributions to the inhibitory activity of MAP kinases.

Docking simulation study and kinase selectivity of f152A1 and its analogs.

f152A1 is a potent inhibitor of MAP kinases and TNFα-transcription. When f152A1 and its analogs are assayed against ERK2, MEK1, and MEKK1, these compounds show different inhibition profiles. It is considered that the highly reactive cis-enone moiety and modifications of the 14-membered resorcylic lactone ring may determine their kinase selectivity and potency. In order to clarify the different potencies of these compounds toward MAP kinases, conformational analysis, molecular orbital studies, and docking simulation studies using model structures of ERK2, MEK1, and MEKK1 have been performed. These studies have revealed that (i) ligand binding does not depend on chemical bonding but on molecular interaction (molecular orbital analysis), (ii) the cis-enone moiety of inhibitors is in the range of Michael addition reaction with the Cys166 residue in ERK2 (docking simulation study), and (iii) molecular shape of M1(8) conformations is the best fit for the ATP binding site of kinases. Considering the molecular docking analysis of these inhibitors in these kinases, molecular shape will be most important to their corresponding kinases activities.