Louis S. Kucera

Oncogenic transformation of rat embryo fibroblasts with photoinactivated herpes simplex virus: rapid in vitro cloning of transformed cells.

Summary Rat embryo fibroblasts (REF) were transformed in vitro with photoinactivated herpes simplex virus. Low passage (7 to 10) HSV-transformed rat cells (t-REF-line G) produced multiple tumours in 49% of newborn rats with a latent period of 20 to 24 weeks. An in vitro cloning procedure for transformants in the uncloned t-REF-line G cells produced clonal lines which varied from non-oncogenic to clonal lines producing tumours with shorter latent periods (10 to 14 weeks) compared to uncloned cells. At passage 30, t-REF-line G-clone 1 cells produced rapidly growing tumours in 100% of the newborn rats with a latent period of only 2 to 3 weeks. Tumour cells (RFS 12-22-75) established in culture produced tumours within 2 weeks after subcutaneous (s.c.) inoculation of weanling rats (100% with tumours) and they were transplantable to 100% of inoculated adult rats. Histopathological examination of all tumours produced in newborn, weanling or adult rats revealed large, poorly differentiated malignant fibrosarcomas; metastatic tumours were observed in the lungs of 10 to 20% of newborn rats inoculated s.c. with RSF cells. Approx. 25 to 50% of the clonal transformed or tumour cells synthesized HSV-specific-antigens detected by immunofluorescence. HSV-transformed and tumour cells are resistent to superinfection by the homologous transforming virus. Since the in vitro cloning procedure for transformant cells can readily segregate cells producing clonal lines varying in oncogenic potential, the procedure might have useful application in elucidating HSV oncogenesis.

Synthesis of 2′,3′-Dideoxy- and 3′-Azido-2′,3′-dideoxy-pyridazine Nucleosides as Potential Antiviral Agents

Abstract The synthesis of 4-methoxy-, 4-amino-3-chloro-, and 4-amino-1-(2,3-dideoxy-B-D-glycero-pentofuranosyl)pyridazin-6-one nucleosides, 6,19 and 20 is described. The synthesis of 3,4-dichloropyridazin-6-one (10) was accomplished in 44% overall yield using bromomaleic anhydride (17) as the starting material. The condensation of the silylated base of 10 with the halogenose 12 in the presence of trimethylsilyl triflate as a catalyst afforded a mixture of3,4-dichloro-1-(3,5-di-O-p-toluoyl-2-deoxy-B-D-erythro-pentofuranosyl)pyrridazin-6-one (13) in 67% and its α-anomer 14 in 12% yield, respectively. A series of 3′-sulfonate esters were prepared to explore the synthesis of 3-chloro-4-hydroxy-1-(3-azido-2,3-dideoxy-B-D-erythro-pentofuranosyl) pyridazin-6-one (32) via 6,3-anhydronucleoside analogues. Compounds 15, 19 and 20 were evaluated against human immunodeficiency virus, human cytomegalovirus, and herpes simplex virus type 1 but were inactive.

Pulmonary surfactant phospholipids modulate priming of rabbit alveolar macrophages for oxidative responses.

We investigated the effect of individual phospholipids contained in pulmonary surfactant (PS) on the macrophage‐activating factor (MAF)‐induced priming of rabbit alveolar macrophages (AMs) for oxidative responses elicited by phorbol myristate acetate (PMA) or opsonized zymosan (Op‐Zym). AMs were incubated with MAF with or without phospholipids for 18 h. After incubation, oxidative responses were elicited with PMA (0.5 μg/ml) or Op‐Zym (250 μg/ml) and monitored by chemiluminescence (GL) assays. The data indicate that natural surfactant inhibited MAF‐induced priming of rabbit AMs for PMA‐ or Op‐Zym‐elicited oxidative responses. Artificial surfactant inhibited PMA‐elicited CL responses but enhanced Op‐Zym‐elicited GL responses. Individual phospholipids differed in modulative activities. Dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylglycerol (DPPG), and phosphatidylinositol (PI) inhibited MAF‐induced priming when the oxidative responses were elicited with PMA. Whereas DPPG inhibited Op‐Zym‐elicited oxidative responses, dipalmitoyl phosphatidylcholine (DPPC) and DOPC primed AMs for increased Op‐Zym‐elicited oxidative responses. DOPC did not affect the binding of phorbol dibutyrate to AMs, which suggests that reduced cell binding of phorbol ester was not responsible for the inhibition of PMA‐elicited oxidative responses in AMs treated with DOPC. Similarly, DPPC, DOPC, and DPPG did not affect the number of zymosan particles phagocytosed by AMs compared to the control, which suggested that enhanced or reduced Op‐Zym‐elicited oxidative responses by phospholipids were not due to altered phagocytic activity of AMs. In conclusion, our data indicate that individual surfactant phospholipid differently modulates priming of AMs for oxidative responses, and the effect of individual phospholipids does not account for the effect of complete PS on priming of AMs.

Phospholipid analogs against HIV-1 infection and disease

Phospholipid analogs are a new class of compounds with potent activity against HIV infection when used alone or conjugated with other therapeutic agents. When conjugated to the nucleoside analog AZT, the resulting phospholipid-AZT conjugate can double target the virus replication cycle by inhibiting the viral reverse transcriptase (by AZT) and inducing the production of defective virus particles that lack functional gp120 expression on the virus surface resulting in reduced capacity to bind to CD4+ cells and inhibition of infected cell-cell fusion (by phospholipid). Of great interest are data indicating that selected phospholipids are active against drug resistant variants, a current major problem in treating HIV/AIDS and controlling the epidemic occurring in various parts of the world. The purpose of this review is to provide current information on the design and synthesis of various types of phospholipids and phospholipid conjugates, in-vitro and in-vivo antiviral activity, tissue distribution, intracellular metabolism, and mechanism of action. The future development of this novel class of compounds offers an exciting approach for reducing the toxicity and enhancing the distribution of therapeutic drugs to the lymphatics and central nervous system and suppressing the emergence of drug resistant variants of HIV.

Boronic Acid Derivatives Targeting HIV-1

A series of novel boronic acid derivatives containing either a pyrimidine or purine base was synthesized. The preparation involved the condensation of 4-bromobutyl boronic acid with the appropriate base. These acyclic nucleosides were designed as potential antiviral agents especially targeting the human immunodeficiency virus. Two analogues, 6-chloro-9-(4-dihydroxyborylbutyl)purine and 2,6-dichloro-9-(4-dihydroxyborylbutyl)purine, exhibited EC50 values of 7.7 μM and 0.99 μM, respectively, in an HIV-1 syncytial plaque reduction assay.

In vitro evaluation and characterization of newly designed alkylamidophospholipid analogues as anti-human immunodeficiency virus type 1 agents

Our laboratories first reported two novel classes of complex synthetic lipids, including alkylamidophosphocholines (PC lipid; CP-51) and alkylamidophosphate ester-linked lipid–AZT conjugates (lipid–AZT conjugates; CP-92), with selective and potent activity against human immunodeficiency virus type 1 (HIV-1). To extend these observations, we synthesized additional PC lipids and lipid–AZT conjugates (INK and INK–AZT conjugate) to evaluate their structure–activity relationships by testing for selectivity against infectious wild-type (wt) and drug-resistant HIV-1 replication, virus fusogenic activity and toxicity for mouse bone marrow cells. PC lipid compounds with medium chain lengths at positions 1 and 2 gave an improved selective index (SI). INK-3, with 12 and 8 carbons and INK-15, with 10 and 12 carbons were among the most selective when evaluated in CEM-SS cells. INK-14, a lipid–AZT conjugate where AZT replaced the choline in PC lipid INK-3, gave the highest SI of >1250 against both infectious wt HIV-1 replication in CEM-SS cells and a clinical isolate in peripheral blood leukocytes. Notably, the PC lipid compounds INK-3 and INK-15, but not the lipid–AZT conjugate INK-14, were potent inhibitors of matched pairs of AZT-sensitive and AZT-resistant HIV-1 clinical isolates. INK-3 also inhibited replication of HIV-2 and TIBO-resistant HIV-1, and inhibited HIV-1-mediated fusogenic activity by 78, 41 and 9% in a dose-dependent manner. The TC50 for mouse bone marrow cells was >100 μg/ml for INK-3 compared to 9.15–14.17 μg/ml for CP-51 and 0.142–0.259 μg/ml for AZT. These data suggest that optimum PC lipid compounds are significantly less toxic than AZT and have high potential as novel therapeutic agents for AIDS.

Lipid synthesis in cultured human embryonic fibroblasts.

We describe here the pathways by which human embryonic fibroblasts synthesize lipids. In these studies, we quantitated the phospholipids by their phosphorus content and by their acyl components. These determinations defined both the chemical composition of the cellular membranes as well as their metabolic turnover. Using radiolabeled precursors, we have shown (a) synthesis of the glycerol moiety via glycolysis and the action of glycerokinase, (b) utilization of both exogenously added and endogenously synthesized fatty acids, (c) synthesis de novo of phosphatidyl choline and phosphatidyl ethanolamine from their base precursors, and (d) the methylation of phosphatidyl ethanolamine yielding phosphatidyl choline. Dividing cells synthesized phosphoglyceride more rapidly than cells in the stationary phase. However, considerable turnover of cellular lipid did occur in the stationary phase.

Priming of rabbit alveolar macrophages for enhanced oxidative responses by herpes simplex virus type 2 infection

The effect of herpes simplex virus type 2 (HSV‐2) infection on the oxidative response in infant and adult rabbit alveolar macrophages (AM) was studied using either phorbol myristate acetate (0.5 μg PMA/ml) or latex (250 μg/ml) as eliciting agents in a chemiluminescence (CL) assay. Results indicated that uninfected infant AM responded to a latex‐elicited but not PMA‐elicited CL response. HSV‐2 infection (moi=1.0) of infant AM for 2 hr at 37° did not alter the PMA or latex‐elicited CL responses. In contrast, uninfected adult AM exhibited a markedly increased CL response when elicited with either PMA or latex. HSV‐2 infection (moi=1) of adult AM for 2 hr further increased both PMA‐ and latex‐elicited CL responses. Increasing the moi to 10 inhibited both PMA‐ and latex‐elicited CL responses. Incubation of uninfected control and HSV‐2 infected adult AM for 18 hr at 37° resulted in spontaneous priming of the cells for increased CL responses. In the absence of PMA HSV‐2 alone failed to elicit a CL response in adult AM. Infection with heat‐inactivated HSV‐2 (moi=1.0 before heat inactivation) did not prime adult AM for enhanced CL responses. AM from BCG immunized adult rabbit produced a considerably higher level CL response that nonimmunized AM; however, HSV‐2 infection of these cells did not further enhance the response. In summary, these data indicate that adult AM but not infant AM can be primed by active HSV‐2 infection for an increased CL response elicited by either PMA or latex.

12–O–Tetradecanoyl–Phorbol–13–Acetate Enhancement of the Tumorigenic Potential of Herpes Simplex Virus Type 2 Transformed Cells

The consequences of tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) interaction with clonal HSV-2-transformed nontumorigenic and weakly tumorigenic cells were studied. Enhancement of anchorage-independent growth (cloning efficiency) occurred in a dose-dependent manner when transformed but not nontransformed cells were pretreated with TPA. All subclonal nontumorigenic cell lines progressed to become tumorigenic after 20 serial passages in the presence of TPA. Also, subclonal, weakly tumorigenic cell lines were enhanced in tumorigenic potential (measured by reduced latent period for tumor formation) after transient treatment with TPA. In summary, TPA irreversibly enhanced the cloning efficiency and tumorigenic potential of transformed cells long after initiation by the virus.

Stimulation of human cell DNA synthesis by defective herpes simplex virus type 2.

Abstract To elucidate herpes simplex virus type 2 (HSV-2) stimulation of cellular DNA synthesis, human embryonic fibroblast (HEF) cells were made quiescent in DNA synthesis by treatment with low serum medium before infection at 42°. Results indicated by that HSV-2 stimulated at least two cycles of cellular DNA synthesis. Exposure of HSV-2 to ultraviolet (uv) light for 30 sec inactivated the ability of virus to expand intracellular [ 3 H]TdR pools; however, the ability of virus to stimulate cellular DNA synthesis was increased. Longer exposures to uv (120 sec) abolished virus stimulation of cellular DNA synthesis. Stocks enriched for defective virus by serial undiluted passage stimulated a significantly higher amount of [ 3 H]TdR incorporation into cellular DNA than standard virus stocks. Data from combined autoradiography and immunofluorescence experiments indicated that during the first cycle of stimulation 30% of the abortively infected cells synthesized viral antigens; about 1% of these cells was simultaneously stimulated in cellular DNA synthesis. Bromodeoxyuridine density-labeling experiments revealed that between 0 and 24 hr HSV-2 stimulated predominantly semiconservative and some repair synthesis of cellular DNA; between 48 and 72 hr virus stimulated only semiconservative cellular DNA synthesis. In conclusion, stimulation of cellular DNA synthesis required viral gene expression and was most likely mediated by defective HSV-2 present in standard virus stocks.