Mutational activation of the Ras oncogene products (H-Ras, K-Ras, and N-Ras) is frequently observed in human cancers, making them promising anticancer drug targets. Nonetheless, no effective strategy has been available for the development of Ras inhibitors, partly owing to the absence of well-defined surface pockets suitable for drug binding. Only recently, such pockets have been found in the crystal structures of a unique conformation of Ras⋅GTP. Here we report the successful development of small-molecule Ras inhibitors by an in silico screen targeting a pocket found in the crystal structure of M-Ras⋅GTP carrying an H-Ras–type substitution P40D. The selected compound Kobe0065 and its analog Kobe2602 exhibit inhibitory activity toward H-Ras⋅GTP-c-Raf-1 binding both in vivo and in vitro. They effectively inhibit both anchorage-dependent and -independent growth and induce apoptosis of H- ras G12V –transformed NIH 3T3 cells, which is accompanied by down-regulation of downstream molecules such as MEK/ERK, Akt, and RalA as well as an upstream molecule, Son of sevenless. Moreover, they exhibit antitumor activity on a xenograft of human colon carcinoma SW480 cells carrying the K-ras G12V gene by oral administration. The NMR structure of a complex of the compound with H-Ras⋅GTP T35S , exclusively adopting the unique conformation, confirms its insertion into one of the surface pockets and provides a molecular basis for binding inhibition toward multiple Ras⋅GTP-interacting molecules. This study proves the effectiveness of our strategy for structure-based drug design to target Ras⋅GTP, and the resulting Kobe0065-family compounds may serve as a scaffold for the development of Ras inhibitors with higher potency and specificity.

Even though elderly populations lack visible or other clinical signs of inflammation, their serum cytokine and C reactive protein levels typically are elevated. However, the origin of age-associated systemic inflammation is unknown. Our previous studies showed that abnormalities in epidermal function provoke cutaneous inflammation, and because intrinsically aged skin displays compromised permeability barrier homeostasis, as well as reduced stratum corneum hydration, we hypothesized here that epidermal dysfunction could contribute to the elevations in serum cytokines in the elderly. Our results show first that acute disruption of the epidermal permeability barrier in young mice led not only to a rapid increase in cutaneous cytokine mRNA expression, but also an increase in serum cytokine levels. Second, cytokine levels in both the skin and serum increased in otherwise normal, aged mice (>12 months). Third, expression of TNFα and amyloid A mRNA levels increased in the epidermis, but not in the liver, in parallel with significant elevations in serum levels of cytokines. Fourth, disruption of the permeability barrier induced similar elevations in epidermal and serum cytokine levels in normal and athymic mice, suggesting the T cells play a negligible role in the elevations in cutaneous and serum inflammatory cytokines induced by epidermal dysfunction. Fifth, correction of epidermal function significantly reduced cytokine levels not only in the skin, but also in the serum of aged mice. Together, these results indicate that the sustained abnormalities in epidermal function in chronologically aged skin contribute to the elevated serum levels of inflammatory cytokines, potentially predisposing the elderly to the subsequent development or exacerbation of chronic inflammatory disorders.

Background While increased levels of circulating inflammatory cytokines in chronologically aged humans have been linked to the development of ageing‐associated chronic disorders (e.g., cardiovascular disease, type II diabetes, osteoporosis and Alzheimer's disease), approaches that reduce circulating cytokines are not yet available. In chronologically aged mice, we recently demonstrated that epidermal dysfunction largely accounts for age‐associated elevations in circulating cytokine levels, and that improving epidermal function reduced circulating cytokine levels. Objective We performed a pilot study to determine whether improving epidermal function reduces circulating pro‐inflammatory cytokine levels in aged humans. Methods Thirty‐three aged humans were topically treated twice‐daily for 30 days, with ≈ 3 mL of an emollient, previously shown to improve epidermal function, while untreated, aged humans and a cohort of young volunteers served as controls. Changes in epidermal function and levels of three key, age‐related, plasma cytokines (IL‐1β, IL‐6 and TNFα) were measured at baseline and after treatment, using Luminex 200™ system. Results We also found significantly higher baseline levels of IL‐1β, IL‐6 and TNFα in aged vs. young humans (P < 0.001), as previously reported. Topical applications of the barrier repair emollient significantly enhanced epidermal permeability barrier function (P < 0.01) and stratum corneum hydration (P < 0.05). In parallel, circulating levels of IL‐1β and IL‐6 normalized, while TNFα levels declined substantially. Conclusion The results of this preliminary study suggest that a larger clinical trial should be performed to confirm whether improving epidermal function also can reduce circulating pro‐inflammatory cytokine levels in aged humans, while also possibly attenuating the downstream development of chronic inflammatory disorders in the aged humans.

In two-stage skin chemical carcinogenesis, phorbol ester 12-Otetradecanoylphorbol-13-acetate (TPA) acts as a promoter essential for clonal expansion of the initiated cells carrying the activated ras oncogenes. Although protein kinase C (PKC) isozymes are the main targets of TPA, their role in tumor promotion remains controversial. We previously reported that mice lacking a Ras/Rap effector phospholipase CE (PLCe À/À mice) exhibited marked resistance to tumor formation in the two-stage skin carcinogenesis. PLCe À/À mice also failed to exhibit basal layer cell proliferation and epidermal hyperplasia induced by TPA, suggesting a role of PLCE in tumor promotion. Here, we show that PLCe À/À mice exhibit resistance to TPA-induced skin inflammation as assessed by reduction in edema, granulocyte infiltration, and expression of a proinflammatory cytokine, interleukin-1A (IL-1A). On the other hand, the proliferative potentials of keratinocytes or dermal fibroblasts in culture remain unaffected by the PLCe background, suggesting that the PLCE's role in tumor promotion may be ascribed to augmentation of inflammatory responses. In dermal fibroblast primary culture, TPA can induce activation of the PLCE lipase activity, which leads to the induction of IL-1A expression. Experiments using small interfering RNA-mediated knockdown indicate that this activation is mediated by Rap1, which is activated by a TPA-responsive guanine nucleotide exchange factor RasGRP3. Moreover, TPA-induced activation of Rap1 and PLCE is inhibited by a PKC inhibitor GF109203X, indicating a crucial role of PKC in signaling from TPA to PLCE. These results imply that two TPA targets, RasGRP3 and PKC, are involved in TPA-induced inflammation through PLCE activation, leading to tumor promotion. [Cancer Res 2008;68(1):64-72]

The transcriptional coactivator complex Mediator facilitates transcription of nuclear hormone receptors and other transcription factors. We have previously isolated the Mediator complex from primary keratinocytes as the vitamin D receptor interacting protein complex. We identified a role for Mediator in keratinocyte proliferation and differentiation in cultured keratinocytes. Here, we investigated the in vivo role of Mediator by generating conditional null mice, where a critical subunit of the Mediator complex, MED1, is deleted from their keratinocytes. The MED1 ablation resulted in aberrant hair differentiation and cycling leading to hair loss. During the first hair follicle cycle, MED1 deletion resulted in a rapid regression of the hair follicles. Hair differentiation was reduced, and β-catenin/vitamin D receptor (VDR) regulated gene expression was dramatically decreased. In the subsequent adult hair cycle, MED1 ablation activated the initiation of hair follicle cycling. Shh signaling was increased, but terminal differentiation was not sufficient. Deletion of MED1 also caused hyper-proliferation of interfollicular epidermal keratinocytes, and increased the expression of epidermal differentiation markers. These results indicate that MED1 plays a critical role in regulating hair/epidermal proliferation and differentiation.