A novel small molecule that directly sensitizes the insulin receptor in vitro and in vivo.

Insulin resistance, an important feature of type 2 diabetes, is manifested as attenuated insulin receptor (IR) signaling in response to insulin binding. A drug that promotes the initiation of IR signaling by enhancing IR autophosphorylation should, therefore, be useful for treating type 2 diabetes. This report describes the effect of a small molecule IR sensitizer, TLK16998, on IR signaling. This compound activated the tyrosine kinase domain of the IR beta-subunit at concentrations of 1 micromol/l or less but had no effect on insulin binding to the IR alpha-subunit even at much higher concentrations. TLK16998 alone had no effect on IR signaling in mouse 3T3-L1 adipocytes but, at concentrations as low as 3.2 micromol/l, enhanced the effects of insulin on the phosphorylation of the IR beta-subunit and IR substrate 1, and on the amount of phosphatidylinositol 3-kinase that coimmunoprecipitated with IRS-1. Phosphopeptide mapping revealed that the effect of TLK16998 on the IR was associated with increased tyrosine phosphorylation of the activation loop of the beta-subunit tyrosine kinase domain. TLK16998 also increased the potency of insulin in stimulating 2-deoxy-D-glucose uptake in 3T3-L1 adipocytes, with a detectable effect at 8 micromol/l and a 10-fold increase at 40 micromol/l. In contrast, only small effects were observed on IGF-1-stimulated 2-deoxy-D-glucose uptake. In diabetic mice, TLK16998, at a dose of 10 mg/kg, lowered blood glucose levels for up to 6 h. These results suggest, therefore, that small nonpeptide molecules that directly sensitize the IR may be useful for treating type 2 diabetes.

A new structural class of proteasome inhibitors that prevent NF-kappa B activation.

The multicatalytic proteinase or proteasome is a highly conserved cellular structure that is responsible for the ATP-dependent proteolysis of many proteins involved in important regulatory cellular processes. We have identified a novel class of inhibitors of the chymotrypsin-like proteolytic activity of the 20S proteasome that exhibit IC50 values ranging from 0.1 to 0.5 microgram/mL (0.1 to 1 microM). In cell proliferation assays, these compounds inhibit growth with an IC50 ranging from 5 to 10 micrograms/mL (10-20 microM). A representative member of this class of inhibitors was tested in other biological assays. CVT-634 (5-methoxy-1-indanone-3-acetyl-leu-D-leu-1-indanylamide) prevented lipopolysaccharide (LPS), tumor necrosis factor (TNF)-, and phorbol ester-induced activation of nuclear factor kappa B (NF-kappa B) in vitro by preventing signal-induced degradation of I kappa B-alpha. In these studies, the I kappa B-alpha that accumulated was hyperphosphorylated, indicating that CVT-634 did not inhibit I kappa B-alpha kinase, the enzyme responsible for signal-induced phosphorylation of I kappa B-alpha. In vivo studies indicated that CVT-634 prevented LPS-induced TNF synthesis in a murine macrophage cell line. In addition, in mice pretreated with CVT-634 at 25 and 50 mg/kg and subsequently treated with LPS, serum TNF levels were significantly lower (225 +/- 59 and 83 +/- 41 pg/mL, respectively) than in those mice that were treated only with LPS (865 +/- 282 pg/mL). These studies suggest that specific inhibition of the chymotrypsin-like activity of the proteasome is sufficient to prevent signal-induced NF-kappa B activation and that the proteasome is a novel target for the identification of agents that may be useful in the treatment of diseases whose etiology is dependent upon the activation of NF-kappa B.

Selective inhibition of the chymotrypsin-like activity of the 20S proteasome by 5-methoxy-1-indanone dipeptide benzamides.

Potent inhibitors of the 20S proteasome that contain a novel indanone head group coupled to di and tripeptides are described. These compounds are the first proteasome inhibitors have demonstrated high selectivity for the chymotrypsin-like activity of the 20S proteasome.

CVT-313, a specific and potent inhibitor of CDK2 that prevents neointimal proliferation.

The activity of cyclin-dependent kinase 2 (CDK2) is essential for progression of cells from G1 to the S phase of the mammalian cell cycle. CVT-313 is a potent CDK2 inhibitor, which was identified from a purine analog library with an IC50 of 0.5 microM in vitro. Inhibition was competitive with respect to ATP (Ki = 95 nM), and selective CVT-313 had no effect on other, nonrelated ATP-dependent serine/threonine kinases. When added to CDK1 or CDK4, a 8.5- and 430-fold higher concentration of CVT-313 was required for half-maximal inhibition of the enzyme activity. In cells exposed to CVT-313, hyperphosphorylation of the retinoblastoma gene product was inhibited, and progression through the cell cycle was arrested at the G1/S boundary. The growth of mouse, rat, and human cells in culture was also inhibited by CVT-313 with the IC50 for growth arrest ranging from 1.25 to 20 microM. To evaluate the effects of CVT-313 in vivo, we tested this agent in a rat carotid artery model of restenosis. A brief intraluminal exposure of CVT-313 to a denuded rat carotid artery resulted in more than 80% inhibition of neointima formation. These observations suggest that CVT-313 is a promising candidate for evaluation in other disease models related to aberrant cell proliferation.

Attenuation of interleukin 2-induced pulmonary vascular leak syndrome by low doses of oral methotrexate.

Pulmonary vascular leak induced in mice by interleukin 2 (IL-2) was attenuated by pretreatment with single or multiple doses of oral methotrexate. Methotrexate also attenuated pulmonary vascular leak when either larger doses of IL-2 or when lymphokine-activated killer (LAK) cells or LAK cells plus IL-2 were administered. Lymphoid infiltrates in the lungs of mice treated with IL-2 and methotrexate were significantly lower. The number of mice surviving treatment with high doses of IL-2 was also significantly increased when these mice were treated with methotrexate. Methotrexate prevented the IL-2-induced increase in the number of splenocytes that were asialo GM1+ but had no effect on Lyt 2+ or L3T4+ cell content. A marginal but significant inhibition in the generation of effector splenocytes that were cytolytic to either YAC or MCA-205 tumor targets was observed in mice treated with methotrexate and IL-2. In vivo studies indicated that methotrexate did not compromise the anti-tumor efficacy of treatment regimens that contained IL-2, LAK cells, or IL-2 and LAK cells. These results demonstrate the potential clinical utility of methotrexate in attenuating pulmonary vascular leak induced by IL-2 without compromising its efficacy. One potential mechanism of action of methotrexate is related to its ability to stimulate the release of adenosine followed by the inhibition of the adhesion of leukocytes to the IL-2-activated endothelium.

Studies evaluating the antitumor activity and toxicity of interleukin-15, a new T cell growth factor: comparison with interleukin-2.

Interleukin-15 is a new cytokine that stimulates the proliferation of T cells and other cells of the immune system. Some of the biological properties of interleukin-15 overlap that of interleukin-2. Using murine models, the present studies have shown that interleukin-15, in vivo, is three to four times more potent than interleukin-2 in generating cytolytic effector splenocytes that lyse YAC target cells. It is approximately one-third as potent as interleukin-2 in inducing specific cytolytic cells that lyse allogeneic target cells. Interleukin-15 is approximately half as potent as interleukin-2 in suppressing pulmonary metastasis induced by MCA-205 tumor cells. The dose of interleukin-15 required to induce pulmonary vascular leak in mice is six times higher than that required for interleukin-2. These results support the view that interleukin-15 exhibits a therapeutic index that is superior to interleukin-2.

A fluorescence assay for geranylgeranyl transferase type I.

A new fluorescence assay for measuring the activity of geranylgeranyl transferase (type I) is described. It does not require the use of either radiolabeled geranylgeranyl diphosphate or the purified recombinant Ras protein substrate with the carboxy terminal sequence of CVLL. Dansyl GCVLL and unlabeled geranylgeranyl diphosphate are used as substrates. The Km for Dansyl GCVLL and for geranylgeranyl diphosphate is 5 microM and 800 nM, respectively. At equimolar concentrations, enzymatic activity is higher when Dansyl GCVLL is used as a substrate compared to Dansyl GCVII. Dansyl GCVLS, a substrate for farnesyl transferase, is inactive in this assay. CVFL is a competitive inhibitor of geranylgeranyl transferase and exhibits a Ki of 200 nM.

HIV type 1 protease activation of NF-kappa B within T lymphoid cells.

NF-kappa B is a nuclear protein of the rel oncogene family capable of enhancing transcription of several cellular genes, including IL-2 and the IL-2 receptor, and viral genes transcribed from the HIV-1 LTR. It has been reported that HIV-1 protease may cleave the NF-kappa B precursor to its active form in vitro. In this study the effects of HIV protease on NF-kappa B precursor activation were examined in Jurkat T cells by introducing a protease expression vector into the cells. Increased NF-kappa B activity was observed and this increased activity was blocked by a specific inhibitor of the viral protease. Viral transcription, as measured using LTR-CAT assays, was only slightly enhanced in the HIV-protease expressing cells, while secretion of IL-2 and expression of the IL-2 receptor were not affected. The limited activation of NF-kappa B by HIV protease appears unlikely to have a significant effect on virus expression or T cell function.

Effect of CL 184,005, a platelet-activating factor antagonist in a murine model of Staphylococcus aureus-induced gram-positive sepsis.

Experiments using a murine model of heat-killed Staphylococcus aureus-induced gram-positive bacterial sepsis indicate that the lethal bacterial effects can be prevented if mice are pretreated with CL 184,005, a platelet-activating factor (PAF) antagonist. CL 184,005 was ineffective when administered after bacterial challenge. Plasma of mice pretreated with CL 184,005 contained significantly less tumor necrosis factor (TNF), suggesting that CL 184,005 interferes with TNF synthesis induced by S. aureus. Spleen-associated TNF protein was also decreased by pretreatment with CL 184,005. Although TNF levels were significantly decreased in mice treated with CL 184,005, interleukin-6 levels in serum were significantly increased. Athymic mice were also susceptible to the lethal effects of S. aureus, suggesting that T cells were not involved. When rats rendered hypotensive with S. aureus were treated with CL 184,005, their blood pressure was normalized. Mice treated with enterotoxin B were not protected if they were pretreated with CL 184,005; however, TNF levels in these mice were significantly lower, suggesting that mediators other than PAF and TNF may contribute to the lethal effects of enterotoxin.

Preclinical studies with platelet-activating factor antagonists in models of septic shock.

Since the isolation and elucidation of the structure of platelet-activating factor (PAF) in the late 1970's, several preclinical studies have suggested that PAF is a key mediator of septic shock induced in animals by either endotoxin or by Gram-negative bacteria. A number of PAF antagonists have been sythesized that protect animals from the lethal effects of endotoxin. Some of these antagonists are in early stages of clinical development. The most advanced cadidate is BN 52021, a ginkgolide, that is in Phase II/III clinical trials in patients with septic shock. Preliminary results with BN 52021 indicate that it is efficacious and significantly reduces mortality associated with Gram-negative sepsis. Pivotal trials with BN 52021 aer ongoing. The present review summarizes the biological effects of PAF and the effect of PAF antagonists in animal models of septic shock. The interrelationship of PAF and tumor necrosis factor (another key mediator of septic shock) is also discussed.

Studies on the effect of CL 306,293, a substituted quinoline carboxylic acid, on the clinical disease induced in mice with LP-BM5 virus.

CL 306,293, a substituted quinoline carboxylic acid, is a potent inhibitor of dihydroorotic acid dehydrogenase, an enzyme essential for the biosynthesis of pyrimidines. In mammalian cell culture, the agent exhibits antiproliferative properties that can be reversed by the addition of uridine. CL 306,293 inhibits the development of the clinical disease in a murine model of immunodeficiency induced by a mixture of LP-BM5 retroviruses. In infected mice, the agent prevents the development of hypergammaglobulinemia, lymphadenopathy, splenomegaly and induction of an IL-2 deficiency. The CD4/CD8 ratio and the number of B cells in the lymph nodes are decreased if the infected animals are treated with CL 306,293. CL 306,293 was more efficacious and potent than 3'-azido-3'-deoxythymidine. The beneficial effects of CL 306,293 observed in this model are most probably related to its antiproliferative properties.

Melanoma cytotoxicity of buthionine sulfoximine (BSO) alone and in combination with 3,4-dihydroxybenzylamine and melphalan.

Buthionine sulfoximine (BSO), a specific inhibitor of glutathione synthesis, showed variable growth-inhibitory activity in different tumor cell lines with a high degree of inhibitory activity against melanoma-derived cell lines. A correlation between BSO growth-inhibitory effects and cellular glutathione peroxidase activity was observed. In contrast, no correlation was demonstrated between the response to BSO and cellular tyrosinase, gamma-glutamylcysteine synthetase, glutathione transferase, gamma-glutamyl transpeptidase, or glutathione reductase activities. BSO enhanced 3,4-dihydroxybenzylamine (3,4-DHBA) (fourfold) and melphalan (threefold) in vitro cytotoxic activity as determined by inhibition of DNA synthesis in human melanoma cells and this enhancement was dependent on the duration of exposure to drug. BSO demonstrated in vivo antitumor activity in B16 melanoma-bearing mice prolonging survival by 29% and in combination with 3,4-DHBA resulted in a slight (48% versus 38%) increase in life span as compared to 3,4-DHBA alone. The combination of BSO and melphalan, however, increased the life span of B16 melanoma-bearing mice by 170%, as compared to melphalan alone (80%). These studies demonstrate a unique in vivo antimelanoma activity of BSO.

Role of ultraviolet radiation in the induction of melanocytic tumors in hairless mice following 7,12-dimethylbenz(a)anthracene application and ultraviolet irradiation.

We examined the role of UVR (UV radiation) (UVA, 320-400 nm; UVB, 290-320 nm; and the combination of UVA and UVB) as a promoter in the induction of cutaneous melanoma. One hundred and seventy hairless mice (Skh-hr2), 6-8 weeks old, were treated in 8 groups: group I, DMBA [7,12-dimethylbenz(a)anthracene] plus UVA; group II, DMBA plus UVA plus UVB; group III, DMBA plus UVB; group IV, DMBA; group V, UVA; group VI, UVA plus UVB; group VII, UVB; group VIII, control. DMBA (0.5% solution) was applied once to promote the formation of dermal melanocytic nevus-like lesions while UVR treatments were conducted 3 times/week for 30 weeks. The mice were examined periodically for the development of multiple pigmented lesions, papillomas, squamous cell carcinomas, melanomas, and lymphomas. Treatment with DMBA plus UVA, DMBA plus UVB, and DMBA plus UVA plus UVB stimulated the development of multiple pigmented nevus-like lesions (85-100%) in mice of groups I, II, III, and IV. Upon necroscopy, 27-33% of animals in groups I, II, and III receiving UVR treatments developed clinically and histologically characterized melanomas. Treatment with DMBA alone did not produce melanomas. DMBA-treated animals in groups I, II, and III which received UVR treatments also developed lymphomas (21-50%). Animals treated with DMBA alone or those that received UVB or the combination of UVB plus UVA (without DMBA) developed only papillomas and squamous cell carcinomas (25-47%). Skin tumors were analyzed for the presence of point mutations in the ras gene. Polymerase chain reaction amplification of DNA and selective oligonucleotide hybridization revealed mutations in the 61st codon of the N-ras gene in the precursor nevus-like lesions and melanoma samples studied. This study suggests that UVR (both UVA and UVB) plays a role as a promoter in the stimulation of melanoma and lymphoma development in hairless mice.

Polyhydroxylated phenylacrylic acid derivatives as new anti-tumor agents.

Preliminary evidence indicates that antitumor agents containing the o-dihydroxybenzene moiety exhibit enhanced antitumor activity toward malignant cells of high oxidative potential, such as melanoma cells. Based on this consideration, 11 hydroxybenzene acrylic acid derivatives of differing redox potential were prepared as potential substrates for the melanoma specific enzyme tyrosinase, that might exhibit general antitumor activity and enhanced cytotoxicity toward melanoma cells. Five of these compounds [alpha-cyano-beta-(4-hydroxyphenyl)-, alpha-cyano-beta-(3,4-dihydroxyphenyl)-, and alpha-cyano-beta-(3,4,5-trihydroxyphenyl)acrylic acid (THPPA), and 3,4-dihydroxy- and 3,4,5-trihydroxybenzalcyanoacetamide] were found to be substrates for tyrosinase with Km values from 0.08 to 4.13 mM and Vmax values from 0.18 to 3.02. These data indicate that as the number of hydroxy groups increases, the rate of oxidation increases, and that cyanoamides were faster reacting than corresponding cyanoacids, with dicyanides the least reactive. In contrast, cyanoamides were less effective as substrates than cyanoacids. In vitro studies showed all but two compounds were active against L1210 (IC50 range 21-980 microM), SK-MEL-28 (IC50 range 54-950 microM), and SK-MEL-30-3 (IC50 range 54-190 microM). Only THPPA was active in vivo against L1210 and B-16 melanoma.

2,4-Dihydroxybenzylamine: a specific inhibitor of glutathione reductase.

The high intracellular level of glutathione is maintained, in part, by the important redox enzyme glutathione reductase. This report describes the properties of a new inhibitor of glutathione reductase, 2,4-dihydroxybenzylamine (2,4-DHBA). The inhibition of glutathione reductase by both 2,4-DHBA and 1,3-bischloroethyl-nitrosourea (BCNU) requires the presence of the co-factor NADPH. However, the inhibition caused by 2,4-DHBA was found to occur much more rapidly. Inhibition of glutathione reductase was time dependent, involved a stoichiometric titration of the enzyme, and was not reversed by gel-filtration indicating an irreversible inhibitory mechanism. The drug interacted at two inhibitory sites as determined by a Hill-type plot analysis. 2,4-DHBA was shown to compete with the substrate oxidized glutathione, and the reducing agents, glutathione and dithioerythritol, were found to protect the enzyme from its inhibitory effect. These results suggest that the inhibition may entail a free radical effect at or near the active site. A structure-activity analysis with other meta-dihydroxybenzene derivatives revealed that the inhibition of glutathione reductase was unique to 2,4-dihydroxybenzylamine.

The mechanism of differential sensitivity to methotrexate of normal and malignant human epidermal cells.

Squamous carcinoma cells are much more sensitive (greater than 10(4) times) to the cytotoxic effects of methotrexate (MTX) and 5-fluorodeoxyuridine (FUDR) than are normal human keratinocytes as measured by cell growth. Among the drugs tested, this phenomenon was found to be specific for MTX and FUDR, since arabinosylcytidine (ARA-C), 13-bis-chloroethylnitrosourea (BCNU), and daunomycin failed to show differences in inhibition between the normal and malignant cell lines. Drug uptake studies did not reveal a significant difference in MTX intracellular levels between malignant and normal epidermal cell lines at 60 min. Thymidine (TdR) salvage was assessed by examining the effects of the presence of 3 microM TdR on MTX-induced cytotoxicity. On the withdrawal of TdR, normal cells demonstrated an increased level of inhibition amounting to 4 orders of magnitude, whereas the squamous-cell carcinoma cells showed no change in sensitivity. Interestingly, the immortal nontumorigenic keratinocyte line (NM-110) was similarly not rescued by the addition of TdR. The high degree of sensitivity TO MTX shown by squamous-cell carcinoma (SCC) and NM-110 cells is attributable to a significant diminution of their ability to use exogenous TdR as compared with that of normal keratinocytes and might be indicative of a biochemical change associated with cellular immortality.

Comparison of cellular protooncogene activation and transformation-related activity of human melanocytes and metastatic melanoma.

Two cell lines (NH and HM1), established from patients with metastatic melanomas, were evaluated for the presence of activated cellular protooncogenes. Northern blot analysis demonstrated increased expression of the c-myc gene (from 9 to 14 times) in NH and HM1 cell lines by densitometric comparison with human melanocyte cell lines. Analysis of the expression of 13 additional cellular protooncogenes revealed either no detectable levels (c-fms, c-abl, v-src, c-erb A1, c-erb B, v-mos, TGF beta, and c-myb) or unaltered expression levels (cH-ras, N-ras, c-fos, and c-sis) in normal human melanocytes and metastatic melanomas. Elevated expression of the c-myc gene was also detected in two long-term cultured melanoma cell lines (RPMI 7951 and SKMEL-30). Analysis of c-myc expression by in situ hybridization in HM1 cells showed that expression was not localized to a sub-population of cycling cells and all cells were overexpressing c-myc mRNA. Differences in relative abundance of c-myc transcripts suggests a relationship with the ability of DNA from these cell lines to efficiently transform NIH 3T3 cells and form colonies on soft agar.

A comparison of the effects of antitumor agents upon normal human epidermal keratinocytes and human squamous cell carcinoma.

The pharmacologic responses of normal and malignant epidermal cells to chemotherapeutic agents were examined in a system which consists of a serum-free "defined" medium. The effects of methotrexate (MTX), fluorodeoxyuridine (FUDR), and hydroxyurea upon cell growth, DNA synthesis, thymidylate synthase activity, and dihydrofolate reductase (DHFR) activity were compared in normal human epidermal keratinocytes (NHEK), newborn epidermal cells (NEC), human squamous cell carcinoma (SCC-25), and a methotrexate-resistant human squamous cell carcinoma (SCC-15R). Normal keratinocytes were found to be 10(3) to 10(4) times less sensitive to the effects of MTX and FUDR than the malignant cells with respect to growth and DNA synthesis. The differential sensitivity to MTX and FUDR was not due to differences in growth media, increased target enzyme activity, e.g., DHFR and thymidylate synthase, or impaired transport of these drugs. The results indicate that the mechanism for the increased sensitivity of the squamous cell carcinoma to MTX and FUDR must involve a process which is distal to the de novo synthesis of dTMP.