Farnesyl-derived inhibitors of ras farnesyl transferase.

The synthesis and evaluation of farnesyl-derived inhibitors of ras farnesyl transferase are presented. Evaluation of inhibitors of farnesyl transferase and comparison with the previously described inhibitor was accomplished using purified enzyme and Amersham's Farnesyl:Transferase enzyme assay kit. These results show an order of magnitude increase in inhibitory activity for beta-ketophosphonic acid over beta-hydroxyphosphonic acid. Incorporation of fluorines in alpha-position, led to an increase in inhibitory activity over the nonfluorinated analogues.

The ribonucleotide reductase inhibitor (E)-2'-fluoromethylene-2'-deoxycytidine (MDL 101,731): a potential topical therapy for herpes simplex virus infection.

The ribonucleotide reductase inhibitor MDL 101,731 was examined for antiviral activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in vitro and in combination with acyclovir in the murine zosteriform model of HSV-1 infection. The in vitro antiviral activity (IC50) for both serotypes of HSV was similar and in the range 23-98 nM for Vero cells. Comparable activities were obtained against acyclovir-resistant viruses. In the zosteriform model, topical combination therapy of MDL 101,731 with acyclovir (5%:5% w/w) applied 48 h after infection was more effective than acyclovir alone and even appeared to promote lesion resolution.

Response of human colon and prostate tumor xenografts to (E)-2'-deoxy-2'-(fluoromethylene) cytidine, an inhibitor of ribonucleotide reductase.

Daily oral or intravenous administration of the ribonucleoside diphosphate reductase inhibitor, (E)-2'-deoxy-2'-(fluoromethylene)cytidine (MDL 101,731), to nude mice caused rapid regression of colon and prostate xenografts. Studies were performed to optimize dosing schedule and route of administration. MDL 101,731 was tested against colon (HT-29) and prostate (PC-3) xenografts using twice weekly oral and intravenous administration. PC-3 tumors regressed almost completely with doses of 20 mg/kg. HT-29 xenografts regressed during intravenous administration of 100 mg/kg MDL 101,731, whereas oral administration was less effective. Based on these data it seems that MDL 101,731 is effective when administered intravenously, twice weekly and is an excellent candidate for clinical development against solid tumors.

Castanospermine analogues: their inhibition of glycoprotein processing alpha-glucosidases from porcine kidney and B16F10 cells.

We have used a simple and efficient procedure for the synthesis of N-5-carboxypentyl-1-deoxynojirimycin, an affinity ligand for alpha-glucosidase I (Bernotas, R. C. and Ganem, B., Biochem. J., 270, 539-540, 1990). The affinity gel was used to purify alpha-glucosidase I in one step from crude extract. In subsequent steps, partially purified alpha-glucosidase II was obtained. We have synthesized several analogues of castanospermine and studied their inhibition of alpha-glucosidase I in vitro using purified alpha-glucosidase I and in vivo in cultured B16F10 cells. Although the castanospermine analogues were significantly less active against the purified enzyme (IC50 approximately 1-23 micrograms/ml) as compared to castanospermine (IC50 = 0.02 microgram/ml), several compounds had up to 30-fold higher activity than castanospermine against alpha-glucosidase I in B16F10 cells, based on the accumulation of G3M7-9N2 oligosaccharide-containing glycoproteins. These results suggest that these analogues with lipophilic side chains cross the membrane barrier more efficiently than castanospermine. Once inside the cell, they may be converted to their active metabolite, castanospermine, by cellular esterases to give enzyme inhibition.

Inhibition of alpha-glucosidase I of the glycoprotein-processing enzymes by 6-O-butanoyl castanospermine (MDL 28,574) and its consequences in human immunodeficiency virus-infected T cells.

The 6-O-butanoyl derivative of castanospermine (MDL 28,574) was previously shown to be approximately 30-fold more potent than the naturally occurring molecule at inhibiting the replication of human immunodeficiency virus (HIV) (D. L. Taylor, P. S. Sunkara, P. S. Liu, M. S. Kang, T. L. Bowlin, and A. S. Tyms, AIDS 5:693-698, 1991). We now report that consistent with its improved anti-HIV activity, MDL 28,574 is more effective (50% inhibitory concentration [IC50], 20 microM) than the parent molecule (IC50, 254 microM) at causing the accumulation of glucosylated oligosaccharides in HIV-infected cells by inhibition of glycoprotein processing. These were predominantly of the glucose 3 type, as determined by P4 Bio-Gel analysis after digestion with purified alpha-glucosidase I, indicating that, intracellularly, this enzyme is the major target for inhibition. MDL 28,574, however, was less active (IC50, 1.27 microM) than castanospermine (IC50, 0.12 microM) against the mutual target enzyme, cellular alpha-glucosidase I, in a cell-free assay system. The increased effects of MDL 28,574 against alpha-glucosidase I in cell culture were attributed to the improved cellular uptake of the more lipophilic derivative. Inhibition of this enzyme activity in HIV-infected H9 cells impaired viral glycoprotein processing and resulted in the expression of abnormally configured gp120. This did not affect virus production, but the virions had decreased infectivity which was partially related to a reduced ability to bind to CD4+ T cells.

6-0-butanoylcastanospermine (MDL 28,574) inhibits glycoprotein processing and the growth of HIVs.

The antiviral activity of 6-0-butanoylcastanospermine (MDL 28,574) [50% inhibitory concentration (IC50: 1.1 microM)] in JM cells infected with a recent isolate of HIV-1 (GB8), was compared with other inhibitors of glycoprotein-processing enzymes. N-butyldeoxynojirimycin (BuDNJ), deoxynojirimycin (DNJ), castanospermine (CAST) or the reverse transcriptase inhibitor 2'3'-dideoxycytidine (ddC) had activities of 56, 560, 29 and 0.1 microM, respectively. MDL 28,574 was at least 50 times more active than BuDNJ and less active but better tolerated in cell culture than ddC, two compounds currently undergoing clinical trials. The CAST derivative showed good protection in H9 cells infected with HIV-1 (RF; IIIB; U455), and HIV-2 (ROD), although the potency was less than that seen in the JM/GB8 system. HIV-1 glycoproteins, gp160 and gp120, synthesized in H9 cells chronically infected with HIV-1 (RF) and treated with MDL 28,574, were characterized by an increase in relative molecular weight of approximately 7-8000 kD. The ratio of gp120 to gp160 was markedly reduced in treated cells and provided further evidence that cleavage of the gp160 precursor molecule is a major consequence of the inhibition of glycoprotein processing. The intracellular target for MDL 28,574 was verified as alpha-glucosidase-I of the processing enzymes by the analysis of high-glucose glycopeptides recovered from treated mouse cells. This activity correlated with the antiviral effect observed against the growth of a mouse retrovirus, Moloney murine leukemia virus (MOLV), in mouse cells.

4',5'-unsaturated 5'-halogenated nucleosides. Mechanism-based and competitive inhibitors of S-adenosyl-L-homocysteine hydrolase.

The design and synthesis of (E)- and (Z)-5'-fluoro-4',5'-didehydro-5'-deoxyadenosine (6 and 13, respectively), a new class of mechanism-based inhibitors of S-adenosyl-L-homocysteine (SAH) hydrolase, is described. A number of analogues of 6 and 13 were synthesized in order to determine the structure-activity relationship necessary for inhibition of the enzyme. Substitution of chlorine for fluorine in 6 (i.e. 44), addition of an extra chlorine to the 5'-vinyl position (i.e. 51 and 52), modification of the 2'-hydroxyl group to the deoxy (34 and 35) and arabino (36 and 37) nucleosides provided competitive inhibitors of SAH hydrolase. Nucleosides 6 and 13, as well as 5'-deoxy-5',5'-difluoroadenosine (14) proved to be time-dependent inhibitors of SAH hydrolase. All three compounds are postulated to inhibit through the potent electrophile derived from oxidation of the 3'-hydroxyl of 6 or 13 to the ketone (i.e. 3 and/or the E-isomer). Consistent with the proposed mechanism of inactivation of SAH hydrolase by 6, 13, and 14 was the observation that incubation of purified rat liver SAH hydrolase with 6 resulted in release of 1 equiv of fluoride ion (by 19F NMR) and incubation with 14 resulted in release of 2 equiv of fluoride ion. The general synthetic route developed for the synthesis of the title nucleosides utilized the fluoro Pummerer reaction for the introduction of fluorine into the requisite precursors. Preliminary antiretroviral data from Moloney leukemia virus (MoLV) is presented and correlates with SAH hydrolase inhibition. Antiviral activity (IC50 against MoLV) ranged from 0.05 to 10 micrograms/mL.

Purification, characterization and antimicrobial properties of an antibiotic (wassumycin) from Streptomyces species 215.

A yellow amorphous compound, wassumycin, was isolated from an unidentified strain of Streptomyces. Wassumycin inhibited the growth of several species of human and animal pathogenic bacteria especially Clostridia. Wassumycin also inhibited the growth of HeLa cells, and showed good antitumour activity against experimental mouse tumours such as L1210, P388 leukaemias, and B16 melanoma. On the basis of the chemical and antimicrobial properties, the antibiotic appeared similar to the chromomycins and olivomycins, but its ultraviolet, infrared, and proton magnetic resonance spectra differed from theirs significantly.

Antitumor activity of a novel synthetic polyamine analogue, N,N'-bis-[3-(ethylamino)-propyl]-1-7-heptane diamine: potentiation by polyamine oxidase inhibitors.

The requirement of the natural polyamines, putrescine, spermidine and spermine, for cell growth suggests that appropriate structural analogues of these compounds could serve as potential antiproliferative agents acting via polyamine antagonism. In this investigation, the antiproliferative activity of N, N'-Bis[3-(ethylamino)-propyl]-1-7-heptane diamine (BEPH), a synthetic polyamine analogue, was investigated employing HeLa cells in culture and L1210 leukemia in mice. BEPH inhibited the growth of HeLa cells with an IC50 of 0.25 microM during a four day culture period. This concentration of the compound was cytotoxic to the cells as evidenced by an 80% reduction in cloning efficiency. Only marginal changes in intracellular polyamine concentrations were observed during incubation with 0.25 microM BEPH. In both HeLa cells and L1210 cells in culture, incorporation of radioactive precursors into DNA, RNA and protein were reduced by BEPH. Inhibition of protein synthesis was discernible prior to inhibition of RNA and DNA in these cells. In mice inoculated i.p. with 10(5) L1210 cells on day 0, i.p. administration of 10.0 mg/kg of BEPH qd(X5) beginning on day 1 prolonged the survival time by 84% compared to controls. The same dose of the compound, in combination with 10.0 mg/kg of N,N'-bis-2-3-butadienylputrescine, an inhibitor of the polyamine catabolizing enzyme polyamine oxidase (PAO), produced a 100% cure rate. Similar results were obtained when BEPH was combined with N-methyl-N'-2-3-butadienylputrescine, another PAO inhibitor. Furthermore, animals cured of the leukemia by the combination chemotherapy were resistant to a subsequent challenge with L1210 cells, indicating the development of tumor "immunity". The striking antitumor activity along with the development of tumor immunity indicate that synthetic polyamine analogues have potential for development as antineoplastic agents.

Effect of polyamine depletion on DNA damage and repair following UV irradiation of HeLa cells.

Treatment of HeLa cells with the polyamine biosynthesis inhibitors, methylglyoxal bis(guanylhydrazone) (MGBG), difluoromethylornithine (DFMO) or a combination of the two, resulted in reduction in cellular polyamine levels. Analysis of UV light-induced DNA damage and repair in these polyamine depleted cells revealed distinct differences in the repair process relative to that seen in cells possessing a normal polyamine complement. Initial yield of thymine dimers and rate of removal of these lesions from cellular DNA appeared normal in polyamine-depleted cells. However, depleted cells exhibited retarded sealing of DNA strand breaks resulting from cellular repair processes, reduced repair synthesis and an increased sensitivity to UV killing. Incision at damaged sites was not affected since ara-C repair-dependent breaks accumulated in a normal fashion. Molecular analysis of inhibited repair sites by exonuclease III and T4 DNA ligase probes suggest that the strand interruptions consist of gaps rather than ligatable nicks, consistent with an interpretation of the repair defect being at the gap-filling stage rather than the ligation step. Observed patterns of differential polyamine depletion by DFMO and MGBG, and partial reversal of repair inhibition by polyamine supplementation, suggests that polyamine depletion per se, rather than some secondary effect of inhibitor treatment, is responsible for the inhibition of repair.

Effects of three irreversible inhibitors of ornithine decarboxylase on macrophage-mediated tumoricidal activity and antitumor activity in B16F1 tumor-bearing mice.

The objective of the present investigation was to compare the effects of three ornithine decarboxylase inhibitors on tumoricidal macrophage and antitumor activities in vivo. alpha-Difluoromethylornithine (DFMO), (2R,5R)-6-heptyne-2,5-diamine, and alpha-(fluoromethyl)dehydroornithine methyl ester (delta MFMOme) were administered continuously in drinking water starting on Day 1 to B16F1 tumor-bearing mice. DFMO, (2R,5R)-6-heptyne-2,5-diamine, and delta MFMOme reduced B16F1 tumor growth, measured on Day 18, up to 87, 79, and 95%, respectively. Similarly, all three ornithine decarboxylase inhibitors reduced B16F1 putrescine and spermidine levels. delta MFMOme was substantially more effective both as an antitumor agent and in reducing polyamines. Both DFMO and delta MFMOme augmented macrophage tumoricidal activity directed against B16F1 target cells. MAP had no effect on macrophage tumoricidal activity. Lipopolysaccharide-stimulated macrophages from delta MFMOme-treated mice also exhibited an increase in interleukin and tumor necrosis factor levels. Furthermore, treatment with a known macrophage activator, gamma-interferon, enhanced the antitumor activity of delta MFMOme. delta MFMOme did not alter natural killer cell activity; however, cytolytic T-lymphocyte induction was reduced by 40 to 50%. These results demonstrate that, in addition to their established antitumor activity, ornithine decarboxylase inhibitors may also potentiate specific tumoricidal effector cell generation in vivo.

Chalcones: a new class of antimitotic agents.

A series of chalcones was evaluated as antimitotic agents. One of these, (E)-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2-methyl-2-pr open- 1-one) (73), was found to be an effective antimitotic agent at a concentration of 4 nM in an in vitro HeLa cell test system. When evaluated in experimental tumor models in vivo, this compound exhibited antitumor activity against L1210 leukemia and B16 melanoma.

Ghilantens: anticoagulant-antimetastatic proteins from the South American leech, Haementeria ghilianii.

Five proteins with anticoagulant and antimetastatic activities were isolated from the salivary glands of the Amazon leech, Haementeria ghilianil. These proteins, designated ghilantens, were co-purified on DEAE-cellulose and heparin-agarose, and were purified by microbore C-18 reverse-phase HPLC. Each variant had a similar molecular weight (18,000), amino acid composition, and a blocked amino terminus. Ghilantens caused a dose-dependent prolongation of the prothrombin time of normal human plasma and blocked the factor Xa-mediated hydrolysis of methoxycarbonyl-D-cyclohexylglycyl-glycl-arginine-p-nitro anillide acetate. Ghilantens were quantitatively absorbed to bovine factor Xa-AffiGel-15 and were eluted with 0.1 mol/L benzamidine, an active-site reversible inhibitor of factor Xa. These findings show that ghilantens can form a reversible association with the enzyme. When administered intravenously to mice by tall vein injection, ghilantens potently suppressed lung metastases of B16-F10 melanoma cells. These findings suggest that ghilantens may have therapeutic value in the treatment of metastatic disease.

Inhibition of glycoprotein processing and HIV replication by castanospermine analogues.

Inhibitors of glycoprotein processing enzymes have been shown to have activity against HIV. Several analogues of the known glucosidase I inhibitor, castanospermine (CAST), were synthesized and evaluated for their inhibitory effect on glucosidases and for antiviral activity against Moloney murine leukemia virus (MOLV) and HIV-1. The most effective analogue was 6-O-butanoyl CAST (B-CAST, MDL 28,574) with an IC50 of 0.05 micrograms/mL against MOLV. A correlation between inhibition of glucosidase I and MOLV replication was observed. This analogue was further evaluated against HIV-induced syncytial formation in HeLa T4+ cells and against productive infection in JM cells infected with HIV 1 (GB8 strain). B-CAST showed an IC50 of 0.3 micrograms/mL in the HeLa T4+ assay, compared to CAST at 11 micrograms/mL. The compound also was more potent (IC50:0.15 micrograms/mL) than CAST (4-6 micrograms/mL) in JM cells. The antiretroviral activity of B-CAST was further confirmed in Friend leukemia virus (FLV) infection in mice. B-CAST showed equivalent activity to AZT and was more potent than CAST in inhibiting FLV-induced splenomegaly in mice. The data presented herein suggest the potential of these novel glucosidase inhibitors as anti-HIV agents.

Potentiation of human lymphokine-activated killer cell activity by swainsonine, an inhibitor of glycoprotein processing.

Interleukin 2 (IL-2) is a secreted glycoprotein which acts as an activation and proliferative signal for lymphocytes expressing membrane-bound glycoprotein IL-2 receptors. We have recently established that swainsonine (SW), an inhibitor of mannosidase II during N-linked glycoprotein processing, augmented mitogen-induced mononuclear leukocyte IL-2 receptor expression and IL-2-induced proliferation. The objective of the present investigation was to examine the effect of SW on lymphokine-activated killer (LAK) cell induction. Human mononuclear leukocytes were treated with various concentrations of SW (0.1-10 micrograms/ml) and IL-2 (1-100 units/ml) for up to 72 h. SW augmented IL-2-induced LAK activity directed against human lung carcinoma, melanoma, and leukemia cells 2-3-fold. LAK activity generated in the presence of SW at suboptimal doses of IL-2 (10 units/ml) was similar to that observed with higher concentrations of IL-2 (100 units/ml) alone. SW treatment alone or in combination with IL-2 increased the percentage of IL-2 receptor-positive cells. Furthermore, pretreatment with SW subsequently enhanced IL-2-induced lymphocyte proliferation. SW-treated mononuclear leukocytes exhibited an increase in high-mannose type glycoproteins based upon [3H]mannose labeling, susceptibility to alpha-mannosidase, and binding to concanavalin A-Sepharose. These results indicate that modulators of glycoprotein processing may be useful in lowering the concentrations of IL-2 required for LAK induction and maintenance.

A simple and rapid microplate assay for glycoprotein-processing glycosidases.

A simple and convenient microplate assay for glycosidases involved in the glycoprotein-processing reactions is described. The assay is based on specific binding of high-mannose-type oligosaccharide substrates to concanavalin A-Sepharose, while monosaccharides liberated by enzymatic hydrolysis do not bind to concanavalin A-Sepharose. By the use of radiolabeled substrates [( 3H]glucose for glucosidases and [3H]mannose for mannosidases), the radioactivity in the liberated monosaccharides can be determined as a measure of the enzymatic activity. This principle was employed earlier for developing assays for glycosidases previously reported (B. Saunier et al. (1982) J. Biol. Chem. 257, 14155-14161; T. Szumilo and A. D. Elbein (1985) Anal. Biochem. 151, 32-40). These authors have reported the separation of substrate from the product by concanavalin A-Sepharose column chromatography. This procedure is handicapped by the fact that it cannot be used for a large number of samples and is time consuming. We have simplified this procedure and adapted it to the use of a microplate (96-well plate). This would help in processing a large number of samples in a short time. In this report we show that the assay is comparable to the column assay previously reported. It is linear with time and enzyme concentration and shows expected kinetics with castanospermine, a known inhibitor of alpha-glucosidase I.

Synthesis of acyclonucleoside hydroxamic acids as inhibitors of ribonucleotide reductase.

N-Hydroxy-alpha-(2-hydroxyethoxy)-1(2H)-pyrimidineacetamides 1-3 were synthesized as potential antitumor agents whose mechanism of action would involve inhibition of ribonucleoside diphosphate reductase (RDPR, EC 1.17.4.1). Acyclonucleoside esters 6-8 were prepared by the stannic chloride catalyzed reaction of methyl chloro[2-(phenylmethoxy)ethoxy]acetate (5) with various silylated pyrimidines, generated in situ from the bases and bis(trimethylsilyl)acetamide. Catalytic didebenzylation of hydroxamate 11 gave 1, while 2 and 3 were synthesized by the reaction of lactones 14 and 22, respectively, with hydroxylamine. In vitro acyclonucleoside hydroxamic acids 1-3 were 3-10-fold less potent than hydroxyurea against calf thymus cytidine diphosphate reductase. 5-Fluorouracil derivative 2 is nearly equipotent with hydroxyurea in inhibiting the growth of HeLa cells, while 1 is a much weaker inhibitor and cytidine derivative 3 is devoid of activity at 200 micrograms/mL.

Effect of murine alpha-, beta-, and gamma-interferons in combination with alpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis, on the tumor growth and metastasis of B16 melanoma and Lewis lung carcinoma in mice.

We have previously established that type I interferon (IFN), a mixture of alpha- and beta-IFN, augments the antitumor activity of alpha-difluoromethylornithine (DFMO), an inhibitor of polyamine biosynthesis, against B16 melanoma. The objective of the present investigation was to extend these earlier observations to metastatic Lewis lung carcinoma and to determine specifically which component(s) of type I IFN potentiates the antitumor activity of DFMO. Furthermore, we wanted to determine whether type II (gamma) IFN can also potentiate the antitumor activity of DFMO. Treatment of animals bearing Lewis lung carcinoma with DFMO, 2% in drinking water (3 g/kg/day), or IFN-alpha/beta (1000 units/mouse) given subcutaneously on alternate days for a total of ten doses alone resulted in 42 and 5% inhibition of tumor growth, respectively. A combination of DFMO and interferon brought about complete elimination of tumors in 12 of the 18 animals, and 94% inhibition of tumor growth in the remainder. DFMO or type I IFN administered alone caused 94 and 26% inhibition of metastasis, respectively. Combination treatment with these two agents resulted in complete elimination of visible metastases. Treatment of mice bearing B16 melanoma with DFMO resulted in 81% inhibition of tumor growth compared to controls. The administration of interferons alone resulted in tumor growth inhibition of 15, 3, 1, and 43% for type I, alpha-, beta-, and gamma-interferons, respectively. Treatment of animals with combination of DFMO and various interferons resulted in inhibition of 94, 93, 86, and 94% of B16 tumor growth for type I, alpha-, beta-, and gamma-interferons, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Antitumor activity of norspermidine, a structural homologue of the natural polyamine spermidine.

The structural specificities of the natural polyamines putrescine (Put), spermidine (Spd) and spermine (Spm) for cell growth are rather stringent, suggesting that appropriate structural analogues of these polycations could serve as potential antineoplastic agents via polyamine antagonism. Norspermidine (Nspd), a homologue of spermidine, had significant antitumor activity against L1210 leukemia, 3LL carcinoma and EL4 lymphoma in mice. The observed antitumor activity of the compound was potentiated by administration of a - difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase. DFMO treatment alone, or in combination with Nspd reduced tumoral Put and Spd levels by greater than 50% in all three tumor models. In animals receiving both Nspd and DFMO, Nspd accumulation in the tumor cells was increased by 50% or more compared to cells from animals receiving Nspd only. Co-administration of Spd, but not Put, abolished the antitumor activity of L1210 observed with DFMO and Nspd treatment, and also reduced the tumoral accumulation of Nspd. These results indicate that appropriate structural analogues of the natural polyamines may be useful as antineoplastic agents.