Guy Hewlett

New helicase-primase inhibitors as drug candidates for the treatment of herpes simplex disease

The vast majority of the world population is infected with at least one member of the human herpesvirus family. Herpes simplex virus (HSV) infections are the cause of cold sores and genital herpes as well as life-threatening or sight-impairing disease mainly in immunocompromized patients, pregnant women and newborns. Since the milestone development in the late 1970s of acyclovir (Zovirax), a nucleosidic inhibitor of the herpes DNA polymerase, no new non-nucleosidic anti-herpes drugs have been introduced. Here we report new inhibitors of the HSV helicase-primase with potent in vitro anti-herpes activity, a novel mechanism of action, a low resistance rate and superior efficacy against HSV in animal models. BAY 57-1293 (N-[5-(aminosulfonyl)-4-methyl-1,3-thiazol-2-yl]-N-methyl-2-[4-(2-pyridinyl)phenyl]acetamide), a well-tolerated member of this class of compounds, significantly reduces time to healing, prevents rebound of disease after cessation of treatment and, most importantly, reduces frequency and severity of recurrent disease. Thus, this class of drugs has significant potential for the treatment of HSV disease in humans, including those resistant to current medications.

The Novel Anticytomegalovirus Compound AIC246 (Letermovir) Inhibits Human Cytomegalovirus Replication through a Specific Antiviral Mechanism That Involves the Viral Terminase

ABSTRACT Human cytomegalovirus (HCMV) remains the leading viral cause of birth defects and life-threatening disease in transplant recipients. All approved antiviral drugs target the viral DNA polymerase and are associated with severe toxicity issues and the emergence of drug resistance. Attempts to discover improved anti-HCMV drugs led to the identification of the small-molecular-weight compound AIC246 (Letermovir). AIC246 exhibits outstanding anti-HCMV activity in vitro and in vivo and currently is undergoing a clinical phase IIb trial. The initial mode-of-action studies suggested that the drug acts late in the HCMV replication cycle via a mechanism distinct from that of polymerase inhibitors. Here, we extend our mode-of-action analyses and report that AIC246 blocks viral replication without inhibiting the synthesis of progeny HCMV DNA or viral proteins. The genotyping of mutant viruses that escaped AIC246 inhibition uncovered distinct point mutations in the UL56 subunit of the viral terminase complex. Marker transfer analyses confirmed that these mutations were sufficient to mediate AIC246 resistance. The mapping of drug resistance to open reading frame UL56 suggests that viral DNA processing and/or packaging is targeted by AIC246. In line with this, we demonstrate that AIC246 affects the formation of proper unit-length genomes from viral DNA concatemers and interferes with virion maturation. However, since AIC246-resistant viruses do not exhibit cross-resistance to previously published terminase inhibitors, our data suggest that AIC246 interferes with HCMV DNA cleavage/packaging via a molecular mechanism that is distinct from that of other compound classes known to target the viral terminase.

In Vitro and In Vivo Activities of the Novel Anticytomegalovirus Compound AIC246

ABSTRACT Human cytomegalovirus (HCMV) remains a serious threat for immunocompromised individuals, including transplant recipients and newborns. To date, all drugs licensed for the treatment of HCMV infection and disease target the viral DNA polymerase. Although these drugs are effective, several drawbacks are associated with their use, including toxicity and emergence of drug resistance. Hence, new and improved antivirals with novel molecular targets are urgently needed. Here we report on the antiviral properties of AIC246, a representative of a novel class of low-molecular-weight compounds that is currently undergoing clinical phase II studies. The anti-HCMV activity of AIC246 was evaluated in vitro and in vivo using various cell culture assays and an engineered mouse xenograft model. In addition, antiviral properties of the drug were characterized in comparison to the current gold standard ganciclovir. We demonstrate that AIC246 exhibits excellent in vitro inhibitory activity against HCMV laboratory strains and clinical isolates, retains activity against ganciclovir-resistant viruses, is well tolerated in different cell types (median selectivity index, 18,000), and exerts a potent in vivo efficacy in a mouse xenograft model. Moreover, we show that the antiviral block induced by AIC246 is reversible and the efficacy of the drug is not significantly affected by cell culture variations such as cell type or multiplicity of infection. Finally, initial mode-of-action analyses reveal that AIC246 targets a process in the viral replication cycle that occurs later than DNA synthesis. Thus, AIC246 acts via a mode of action that differs from that of polymerase inhibitors like ganciclovir.

A new model for investigations of t-cell functions in mice: differential immunosuppressive effects of two monoclonal anti-thy-1.2 Antibodies.

Two monoclonal anti-Thy-1.2 antibodies were investigated for their activity in eliminating T cells in vitro and in vivo. Both antibodies exert a complement-dependent cell cytotoxicity in vitro. Antibody B that belongs to the IgM class shows a 100-fold higher complement-dependent cytotoxic activity than antibody C, which is of IgG2a class. However, administration of antibody C into Balb/c mice results in the elimination of T cells as determined by the failure of different T-cell functions. Within 24 hours after administration of antibody C, the reactivity of spleen of lymph-node cells to T-cell mitogens, the antibody response to the T-cell-dependent antigen SRBC and the SRBC-induced delayed-type hypersensitivity are completely abolished. These effects are dose-dependent in a dose range of 0.1-1.0 mg Ig protein per animal and affects only T cells in the peripheral lymphoid organs. The Thy-1.2-bearing cells residing in the thymus are not impaired by the treatment of the animals with this monoclonal antibody and are able to repopulate the peripheral lymphoid organs within 30 to 60 days. Investigations into the mode of action of the removal of peripheral T cells revealed that antibody-C-coated Thy-1.2-bearing cells are rapidly phagocytosed by macrophages, while antibody-B-coated Thy-1.2-bearing cells are not. This might be the reason for the differential in-vivo activities of the two monoclonal antibodies. A model with new qualities for the study of functions and the regeneration of T cells in vivo has been established.

Strategies for optimising serum-free media

ConclusionTwo of the main reasons for developing serum-free medium are to improve the growth and to increase the productivity of a cell. This review has attetmpted to show that these goals can be achieved far more easily than generally realised by utilising the methods that have been developed during the past decade and by using a limited number of standard supplements. Serum-free media have contributed greatly to the advances made in recent years in the fields of cell biology and mammalian cell technology and it is clear that they will continue to play a major role in the clarification of cellular regulatory processes and in the refinement of processes in modern cyto-technology.

Activation of T-Cells by a Macrophage or 2-Mercaptoethanol Activated Serum Factor is Essential for Induction of a Primary Immune Response to Heterologous Red Cells in vitro

Many immunological phenomena are closely associated with or dependent on the functional activities of M0. This classifies M0 as belonging to the immune system, and the elucidation of the M0 function should allow a better understanding of how thymus-derived (T) and bone marrow-derived (B) lymphocytes respond to antigenic stimulation. Most studies concerning M0 fimction were carried out in vitro because there is no way to eliminate M0 from the whole body; only short time ftmctional blockade is possible. In vitro immune responses seem to reflect in vivo capabilities since both are comparable with regard to kinetics, magnitude, Ig class, immumogenicity

A method for the quantitation of interleukin-2 activity

A bioassay for the determination of interleukin-2 activity is described. We have compared the traditional method of data processing, which involves probit analysis and curve fitting, with a simpler method based on the so-called AUC (area under the curve). The latter method is readily applicable to spreadsheet software and can handle large amounts of data.

Viral Infections and Treatment

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Development of protein-deficient serum-free culture media for adherent cell lines suited for large scale cultivation

Procedures are described for the devlopment of less expensive, protein-deficient serum-free media for the cultivation of mammalian adherent cell lines. Starting with a proteinrich chemically defined, serum-free culture medium, the concentrations of the expensive or highly concentrated proteins, or both, such as bovine serum albumin (BSA), insulin, or transferrin are systematically reduced one after the other until the minimal concentrations are reached at which the cells grow as efficiently as in the initial protein-rich medium formulation. The resulting protein-deficient serum-free media are of interest for an economical, large scale production of natural substances from mammalian cell cultures. The method is described using as an example the human cell line prostate adenocarcinoma. The protein content of a serum-free medium developed for these cells was reduced from originally 1020 mg/l to finally <1 mg/l.

Xenograft Model for Identifying Chemotherapeutic Agents against Papillomaviruses

ABSTRACT The report describes the establishment and characterization of a mouse xenograft transplantation model for the study of papillomavirus infection of bovine skin. Calf scrotal skin was inoculated with bovine papillomavirus type 2 before grafting it to the dorsum of severe combined immunodeficient mice. The grafted skin contained epidermis, dermis, and a thin layer of fat. After 5 months the induced warts not only showed histological features of papillomavirus infections but also tested positive for viral DNA and papillomavirus capsid antigen. The formation of infectious virions was demonstrated by inoculation of new transplants with crude extract from the induced warts as well as in a cell culture focus assay. Topical application of bromovinyl-2′-deoxyuridine led to a reduction in viral DNA content in the developing wart. This small-animal xenograft model should be useful for characterizing antiviral compounds and providing an understanding of the regulation of papillomavirus infections.