Identification of MK-944a: a second clinical candidate from the hydroxylaminepentanamide isostere series of HIV protease inhibitors.

Recent results from human clinical trials have established the critical role of HIV protease inhibitors in the treatment of acquired immune-deficiency syndrome (AIDS). However, the emergence of viral resistance, demanding treatment protocols, and adverse side effects have exposed the urgent need for a second generation of HIV protease inhibitors. The continued exploration of our hydroxylaminepentanamide (HAPA) transition-state isostere series of HIV protease inhibitors, which initially resulted in the identification of Crixivan (indinavir sulfate, MK-639, L-735,524), has now yielded MK-944a (L-756,423). This compound is potent, is selective, and competitively inhibits HIV-1 PR with a K(i) value of 0.049 nM. It stops the spread of the HIV(IIIb)-infected MT4 lymphoid cells at 25.0-50.0 nM, even in the presence of alpha(1) acid glycoprotein, human serum albumin, normal human serum, or fetal bovine serum. MK-944a has a longer half-life in several animal models (rats, dogs, and monkeys) than indinavir sulfate and is currently in advanced human clinical trials.

Non-active site changes elicit broad-based cross-resistance of the HIV-1 protease to inhibitors.

Three high level, cross-resistant variants of the HIV-1 protease have been analyzed for their ability to bind four protease inhibitors approved by the Food and Drug Administration (saquinavir, ritonavir, indinavir, and nelfinavir) as AIDS therapeutics. The loss in binding energy (DeltaDeltaG(b)) going from the wild-type enzyme to mutant enzymes ranges from 2.5 to 4.4 kcal/mol, 40-65% of which is attributed to amino acid substitutions away from the active site of the protease and not in direct contact with the inhibitor. The data suggest that non-active site changes are collectively a major contributor toward engendering resistance against the protease inhibitor and cannot be ignored when considering cross-resistance issues of drugs against the HIV-1 protease.

Elucidation of basic mechanistic and kinetic properties of influenza endonuclease using chemically synthesized RNAs.

Influenza virus utilizes a unique mechanism for initiating the transcription of viral mRNA. The viral transcriptase ribonucleoprotein complex hydrolyzes host cell transcripts containing the cap 1 structure (m7GpppG(2'-OMe)-) to generate a capped primer for viral mRNA transcription. Basic aspects of this viral endonuclease reaction are elucidated in this study through the use of synthetic, radiolabeled RNA substrates and substrate analogs containing the cap 1 structure. Unlike most ribonucleases, this viral endonuclease is shown to catalyze the hydrolysis of the scissile phosphodiester, resulting in 5'-phosphate- and 3'-hydroxyl-containing fragments. Nevertheless, the 2'-OH adjacent to the released ribosyl 3'-OH is shown to be important for catalysis. In addition, while the endonuclease steady-state turnover rate is measured to be 2 h(-1), phosphodiester bond hydrolysis is not rate-limiting. The direct generation of a free 3'-OH and the subsequent slow release of this product are consistent with the viral need for efficient use of the capped primer in subsequent reactions of the influenza transcriptase complex.

Expression and purification of retroviral HIV-1 reverse transcriptase.

Modern molecular biology techniques have provided valuable tools which allow for the expression of large amounts of enzyme in E. coli. For potential therapeutic targets such as HIV-1 reverse transcriptase, it is desirable that the enzyme studied is pure and correlates to the active form of the enzyme found in vivo. This poses a particular challenge for those researchers studying HIV-RT since a significant degree of heterogeneity is introduced by nonspecific proteolytic cleavage of the p66 subunit by E. coli proteases. The advantage of the purification protocol presented here is that the association of monomers is facilitated by mixing an excess of p51 subunit, which is truncated at a site that is N-terminal to known bacterial cleavage sites, with p66 protein. This avoids enzymatic processing of the larger subunit since the formation of heterodimeric RT is rapid and the dimer is stable against proteolytic cleavage. Therefore, it is possible to isolate a pure homogeneous p66/p51 heterodimer. An enzyme prepared in this manner yields crystals that defract to a 3.2-A resolution. It has also been used to study both sensitivity of HIV-1 RT mutants to azidothymidine triphosphate and the kinetics of a potent nonnucleoside RT inhibitor (L-743,726). Finally, it is interesting to note the similarity of HIV-1 RT with reverse transcriptases from other lentiviruses (FIV and EIAV RT). Both of these enzymes consist of heterodimers of p66 and p51 subunits and share other biophysical characteristics. Purification of these reverse transcriptases can, in all likelihood, be optimized by using methods similar to those described in this chapter.

Antitumor effect of deferoxamine on human hepatocellular carcinoma growing in athymic nude mice.

BACKGROUND: Iron is essential for the growth of all living cells. One of the important intracellular roles for iron is in the activation of ribonucleotide reductase, the enzyme that catalyzes the first step in DNA synthesis. Thus, the intracellular iron level may serve as a regulator of cell growth. The authors tested the hypothesis that lowering body iron concentration inhibits the growth of human-derived hepatocellular carcinoma (HCC) cells by depleting these cells of iron. Deferoxamine (DFO), an iron-chelating agent, was used to lower intracellular iron level. METHODS: HCC cells, PLC/PRF/5 (7 x 10(6) cells/mouse), were transplanted subcutaneously into athymic nude mice. When tumors reached 200-300 microliters in size, mice with comparable tumor sizes were paired; one was treated with DFO (300 mg/kg body weight/day, 5 days/week) intraperitoneally while the other received no treatment. RESULTS: Eight pairs of mice with HCC were observed for 5-18 weeks. Mean tumor growth rates (TGR) (mean +/- standard error) for the untreated and treated mice were 30.5 +/- 3.7 microliters/week and 11.9 +/- 1.5 microliters/week. The difference was significant (P < 0.02). In the second set of studies, DFO treatment was begun when the tumor size was smaller (100-200 microliters). Four pairs of mice were observed for 4-15 weeks; mean TGR for the four untreated mice was 18.1 +/- 5.1 microliters/week. In two mice treated with DFO, tumors regressed completely by the seventh week after initiation of treatment. The two remaining mice on DFO therapy had much slower growing tumors, with a mean TGR of 1.8 +/- 0.5 microliters/week. CONCLUSIONS: Thus, our results suggest that (1) reduction of intracellular iron concentration by DFO may be useful as antitumor therapy in HCC and (2) the favorable effects of DFO treatment are best seen when treatment is begun when the tumor is small.

Iron enhances tumor growth. Observation on spontaneous mammary tumors in mice.

Iron is essential for the growth of all cells, including tumor cells. The authors previously reported that a variety of transplantable tumors grew faster and larger in mice that were on an iron-rich diet compared with those on an iron-deficient diet. In this study the authors examined the relationship between iron in the diet and development of tumors in mice that are known to develop spontaneous tumors--C3H/HeN-MTV+(C3H-MTV+) mice that were congenitally infected with mammary tumor virus. These mice have a greater than 96% chance of developing mammary tumors between the ages of 7.2 and 9.2 months. Fifteen C3H-MTV+ weanlings were given a low-iron diet (5 mg iron/kg diet), and 15 were given diets with normal amounts of iron (180 mg Fe/kg diet). Thirteen of the 15 mice from the low-iron group and all 15 mice from the normal-iron group developed tumors. The average tumor growth rate in the normal-iron group was 112%/wk, compared with 62%/wk for the low-iron group. The difference in tumor growth rate between the two groups was significant (P = 0.02 by Student's t test). In this study, low iron intake did not prevent tumor development, but the results confirm the authors' previous report that iron nutrition of the host affects tumor growth; tumors grow better in an iron-rich environment. High levels of iron in the diet may enhance tumor growth, and this should be considered when treating patients with cancer.

Prognostic importance of serum transferrin and ferritin in childhood Hodgkin's disease.

The relationship of iron-binding proteins to prognosis was studied in 50 children at the Children's Hospital of Philadelphia, newly diagnosed with Hodgkin's disease (HD). There were five patients with Stage I, 18 with Stage II, 14 with Stage III, and 13 with Stage IV. Initial serum ferritin, transferrin, iron, hemoglobin (Hb), erythrocyte sedimentation rate (ESR), and A or B symptoms were analyzed for their association with progression-free survival (PFS). There was a linear increase of mean and median ferritin levels and a decrease of mean and median transferrin levels with advancing stages. Also, there was a significant inverse correlation between ferritin and transferrin (P less than 0.001). In univariate analyses, high ferritin (greater than 142 ng/ml) (P = 0.02) and low transferrin (less than or equal to 250 mg/dl) (P = 0.008) were significantly associated with poor PFS. Serum iron, Hb, ESR, and A or B symptoms were not associated with PFS. Stepwise proportional hazards regression analysis of all factors showed that transferrin was the only factor significantly associated with PFS. These preliminary results suggest that serum transferrin can also be used as a prognostic factor in addition to serum ferritin and that it may be helpful to assay both serum ferritin and transferrin as prognostic factors in childhood HD. Further testing of large groups of patients is needed to determine whether they are independent of tumor bulk and other established prognostic factors.

Effect of iron and desferoxamine on cell growth and in vitro ferritin synthesis in human hepatoma cell lines.

To investigate the effects of iron supplementation on hepatoma cell growth, cells from a human hepatoma cell line, PLC/PRF/5, were grown in RPMI 1640 supplemented with 0, 10 and 20 micrograms/ml of FeSO4 and harvested weekly. At the end of 6 wk culture, cell mass measured 9.6, 14.7 and 13.2 gm, respectively. Amounts of ferritin from these cell masses were 0 (undetectable), 0.89 and 2.27 micrograms/gm of cells. To study the effects of iron deprivation of hepatoma cells, three human hepatoma cell lines (PLC/PRF/5, Hep G2 and Hep 3B) were incubated in tissue culture medium mixed with graded amounts of an iron-chelating agent, desferoxamine, for 48 to 96 hr at 37 degrees C with 5% CO2. Over 50% cell death in PLC/PRF/5 cells and 30% to 50% cell death in Hep G2 and Hep 3B cells were observed 48 to 72 hr after exposure to desferoxamine. Addition of ferric citrate partially reversed the cytotoxic effect of desferoxamine. On the other hand, viability of control cells, human diploid cell line (WI 38), was not affected by desferoxamine. Even after 96 hr exposure to desferoxamine, cell death was only 2% to 4%. These results suggest that (a) iron enhances tumor cell growth, (b) iron induces increased ferritin synthesis by tumor cells in vitro and (c) iron depletion causes tumor cell death but has little effect on normal human diploid cells. These findings should be considered when designing treatment of patients with hepatoma. Iron oversupply in patients with cancer might enhance tumor growth and adversely affect cancer therapy. Iron chelation with desferoxamine might have a place in the treatment of patients with hepatoma in conjunction with other anticancer agents.

Effects of isoferritins on human granulocytes.

Serum ferritin levels are often elevated in patients with certain cancers and these elevations are, in part, derived from the tumors. In such patients, the increased levels of serum ferritin are associated with a poor prognosis. This association may be explained in part by biological effects of ferritin on lymphocytes: inhibition of E-rosette formation, masking of cell surfaces and suppression of lymphocytes' response to mitogens in vitro. The authors hypothesized that ferritins from tumor tissues also exert adverse effects on human granulocytes that are involved in tumoricidal activity. Three granulocyte functions were tested: nitroblue tetrazolium test, phagocytosis, and production of hydrogen peroxide. The results supported the authors' hypothesis: NBT reduction and phagocytosis are decreased in granulocytes exposed to ferritins, more so with tumor ferritins, than normal ferritin, and H2O2 production is less in granulocytes previously exposed to ferritins from tumor and nontumor tissues than cells not exposed to ferritins. However, the inhibitory effects of ferritins on H2O2 production can be reversed if granulocytes are further stimulated by phorbol myristate acetate (a membrane stimulant). If the elevated serum ferritin in cancer patients impairs granulocyte functions, in vivo, then it may increase the risk of infection, decrease tumoricidal host responses, and, thereby, contribute to the poor prognosis of these individuals.

Basic and acidic isoferritins in the sera of patients with neuroblastoma.

Serum ferritin frequently is elevated in patients with neuroblastoma. Isoferritins extracted from neuroblastoma tumors and cells in culture show a wide range from basic (rich in L subunit) to acidic (rich in H subunit) isoferritins. Total ferritin is a combination of basic and acidic isoferritins. Forty-four serum samples from 25 patients with neuroblastoma were measured for basic and acidic isoferritin levels by radioimmunoassay using antibodies to liver (basic) ferritin and HeLa (acidic) ferritin. Normal ranges for basic and acidic serum ferritins were 7 to 142 ng/ml (median, 30 ng/ml) and 0 to 12 ng/ml (median, 3.4), respectively. Basic ferritins in the 44 neuroblastoma sera ranged from 0 to 1460 ng/ml, and acidic ferritins, 0 to 40 ng/ml. Sera with high levels of acidic ferritins always had increased basic ferritins. Thus, acidic/basic ferritin ratios were nearly constant, less than 0.3 in all sera. There was a significant linear correlation between basic and acidic isoferritins (r = 0.833). These results suggest that neuroblastoma tumors produce both basic and acidic isoferritins and release them into circulation. However, there is no acidic ferritinemia not accompanied by basic ferritinemia. Therefore, the commercial assay for basic isoferritin currently seems sufficient for clinical prognostic purposes.

Iron nutrition and tumor growth: decreased tumor growth in iron-deficient mice.

Groups of 15 mice of three different laboratory strains (BALB/c, C3H/He, DBA/2) were fed on a low iron diet (5 mg iron/kg diet), and three similar groups of 15 mice were maintained on a normal iron diet (312 mg iron/kg diet). When the low iron diet group became iron deficient, tumor cells (5 x 10(5) cells/mouse) of CA07-A (colon adenocarcinoma), HE129 (hepatoma), and M119 (mammary adenocarcinoma) were inoculated s.c. in BALB/c, C3H/He, and DBA/2 mice, respectively. All mice developed tumors, tumors grew more slowly, and the mean tumor sizes were smaller in the low iron diet group at nearly all weekly observations in all three strains of mice. No apparent differences in the behavior, activity (e.g., movement, climbing, running, grooming, etc.), and appearance were observed between low iron diet and normal iron diet mice. The mean body weight of mice at transplantation was less in the low iron than in the normal iron groups for the BALB/c strain but higher in the low iron groups of C3H/He and DBA/2 mice, indicating that food intake of mice on a low iron diet was not impaired. These results suggest that iron nutrition of the host affects tumor growth; tumor cells grow better in an iron-rich environment. This knowledge should be considered when designing treatment for patients with cancer. Iron oversupply in cancer patients might enhance tumor growth and adversely affect cancer therapy.

Serum ferritin in hepatocellular carcinoma.

The serum ferritin level was detected by radioimmunoassay in 142 patients with hepatocellular carcinoma (HCC). Serum ferritin level was raised (greater than ng/ml) in 38% (54/142) of patients with HCC. There was no difference in serum ferritin levels between stages 1, 2 and 3 HCC. None of the 4 patients with stage 1 HCC had serum ferritin levels above 300 ng/ml. In this small group of patients, measurement of serum ferritin was not a satisfactory indicator of stage 1 HCC. A combination of serum ferritin alpha fetoprotein (AFP) measurements may be useful for a rising suspicion of HCC. We found that a correct diagnosis of HCC was made in 38% (54/142) of patients by measurement of ferritin alone, and in 83.8% (119/142) by measurement of AFP alone, but in 92.3% (131/142) by measurement of a combination of these two markers. Serum ferritin estimation may be helpful in detection of HCC without elevated AFP. Among 12 cases of HCC with serum AFP less than 500 ng/ml, 6 cases (50%) had serum ferritin levels greater than ng/ml. There was no correlation between serum ferritin and AFP, nor between serum ferritin and HBsAg. However, among 12 patients with very high ferritin levels (range 992-3000 ng/ml), 11 (91.7%) had AFP levels of more than 500 ng/ml (mean = 4800 ng/ml, range 500-32000 ng/ml).

Serum selenium assay following serum ferritin assay.

Serum samples from 10 individuals were divided into six subsamples. Three of these were tested for ferritin by radioimmunoassay. All six samples from each subject were then tested for selenium by neutron activation assay. There was no significant difference between the selenium values that had been obtained from the sera that had been tested for ferritin and those values that had been obtained for sera not tested for ferritin.

Source of increased ferritin in neuroblastoma: studies with concanavalin A-sepharose binding.

Serum ferritin is present in two forms--a glycosylated form that results from active secretion by cells and a nonglycosylated form that is directly released by damaged cells. Glycosylated ferritin binds to concanavalin A (Con A) through the glucose and/or mannose residues of the molecule. Patients with neuroblastoma frequently present at diagnosis with abnormally elevated levels of serum ferritin. The ferritin levels will, however, return to normal with clinical remission, suggesting that the tumor is the origin of the elevated ferritin. With the use of a Con A binding assay, an investigation was made as to whether the increased levels of serum ferritin at diagnosis in neuroblastoma patients resulted from active secretion by the tumor or were the consequence of direct release of ferritin from damaged tissue. Serum samples were collected at diagnosis from 36 children with neuroblastoma and from 16 normal healthy subjects. Tissue ferritins were purified from normal human liver, placenta, HeLa cells, human neuroblastoma, and hepatoma cells grown in culture. Serum and tissue ferritins were measured before and after binding with Con A. Sixty-three percent of serum ferritin from neuroblastoma patients and 66% of serum ferritin from normal subjects were bound to Con A, suggesting that they were glycosylated and were likely to have been secreted. On the other hand, only 28% of tissue ferritin were bound to Con A. Furthermore, most patients showed abnormally elevated levels of serum ferritin, and 63% of these ferritins were bound to Con A. These results are compatible with the hypothesis that much of the elevated ferritin in sera of patients with neuroblastoma seen at diagnosis is the result of secretion of ferritin by the tumor.

Serum ferritin as a prognostic indicator in neuroblastoma: biological effects of isoferritins.

These studies suggest that a) the levels of serum ferritin are closely related to the prognosis of patients with neuroblastoma; b) the increased amounts of ferritin in the serum of patients with neuroblastoma are, in part, derived from the tumor; c) isoferritins from neuroblastoma cells exert adverse effects on the host immune response and host defenses; and d) therefore, isoferritins released from tumors may, in part, be responsible for the poor prognosis of patients with elevated levels of serum ferritin.

Human ferritins present in the sera of nude mice transplanted with human neuroblastoma or hepatocellular carcinoma.

Fifteen nude mice were inoculated with a human neuroblastoma cell line and 14 with a human primary hepatocellular carcinoma cell line. Human ferritins were detected in the sera of the mice which developed tumors. Of 14 mice bearing human neuroblastoma, 12 had human liver-type ferritin (8 to 52 ng/ml) in their sera, and three of these also had HeLa-type ferritin (acidic ferritin) (29 to 40 ng/ml). Of 10 nude mice bearing human primary hepatocellular carcinoma, eight had human liver-type ferritin (10 to 820 ng/ml), and one of these had HeLa-type ferritin at a level of 43 ng/ml. Since the ferritins in the sera of these mice were produced by the human tumor cells, these observations support the hypothesis that the elevated ferritins often found in the serum of patients with cancer are, in part, derived from their tumors.