R. Willebrords

In vitro activity of pirodavir (R 77975), a substituted phenoxy-pyridazinamine with broad-spectrum antipicornaviral activity.

Pirodavir (R 77975) is the prototype of a novel class of broad-spectrum antipicornavirus compounds. Although its predecessor, R 61837, a substituted phenyl-pyridazinamine, was effective in inhibiting 80% of 100 serotypes tested (EC80) at concentrations above 32 micrograms/ml, pirodavir inhibits the same percentage of viruses at 0.064 micrograms/ml. Whereas R 61837 was active almost exclusively against rhinovirus serotypes of antiviral group B, pirodavir is broad spectrum in that it is highly active against both group A and group B rhinovirus serotypes. Pirodavir is also effective in inhibiting 16 enteroviruses, with an EC80 of 1.3 micrograms/ml. Susceptible rhinovirus serotypes were rendered noninfectious by direct contact with the antiviral compound. Their infectivity was not restored by dilution of virus-drug complexes, but was regained by organic solvent extraction of the compound for most serotypes. Neutralized viruses became stabilized to acid and heat, strongly suggesting a direct interaction of the compounds with viral capsid proteins. Mutants resistant to R 61837 (up to 85 times the MIC) were shown to bear some cross-resistance (up to 23 times the MIC) to the new compound, indicating that pirodavir also binds into the hydrophobic pocket beneath the canyon floor of rhinoviruses. Pirodavir acts at an early stage of the viral replication cycle (up to 40 min after infection) and reduces the yield of selected rhinoviruses 1,000- to 100,000-fold in a single round of replication. The mode of action appears to be serotype specific, since pirodavir was able to inhibit the adsorption of human rhinovirus 9 but not that of human rhinovirus 1A. Pirodavir is a novel capsid-binding antipicornavirus agent with potent in vitro activity against both group A and group B rhinovirus serotypes.

A comparative test of fifteen compounds against all known human rhinovirus serotypes as a basis for a more rational screening program

A systematic evaluation of 15 rhinovirus capsid-binding agents against all 100 serotyped human rhinoviruses revealed the existence of two virus groups, based upon differential susceptibility to antiviral compounds. Elongated and short-chained compounds preferentially inhibited groups A and B. The positions of the rhinoviruses within a map derived from a multivariate analysis allow for the selection of a panel of 17 rhinoviruses, for which the median antiviral inhibitory value against them will accurately predict the median value against 100 serotypes. This rationalizes the search for broad-spectrum capsid-binding antirhinovirus drugs, or combinations of drugs with complementary spectra that may be necessary to effectively inhibit both type A and type B viruses.

Lack of quantitative correlation between inhibition of replication of rhinoviruses by an antiviral drug and their stabilization.

SummaryR 61 837, a new antirhinovirus compound, was able to protect several susceptible rhinoviruses against inactivation by mild acidification or heat. This observation strengthens the hypothesis that the drug exerts antiviral activity by a direct interaction with the viral protein capsid to stabilize the particle. However, the minimal concentrations necessary to inhibit either acetate or citrate or heat inactivation were different for each of five tested serotypes and we therefore conclude that stabilization and inhibition of replication are not causally linked but parallel events, both independently resulting from the binding of the drug to the viral capsid.Studies using drug resistant mutants of HRV 51 and HRV 9 confirmed this lack of quantitative correlation. The mutants were also shown to be cross resistant to a panel of seven different reference antirhinoviral drugs including SDS, WIN 51711, chalcone, dichloroflavan and MDL 20,610. This indicates that all these compounds bind to the same site corresponding to the hydrophobic pocket within the viral protein VP 1 β-barrel structure of HRV 14.

Influence of the synthetic microtubule inhibitor erbulozole (P.I.N.N.) (R 55 104), a new tubulozole congener, and gamma irradiation on murine tumors in vivo

Erbulozole (P.I.N.N.) (R 55 104) is a more water soluble congener of the synthetic microtubule inhibitor tubulozole (R 46 846) exhibiting a reversible antimicrotubular activity in vitro at a dose (1.56 x 10(-8) M) which is at least 10-fold lower. The compound also has an antiinvasive potential and shows antitumoral effects both in vitro and in vivo when administered appropriately. Eighty mg/kg R 55 104, given orally 6 h before or 3 h after radiotherapy, displays a prominent interactive effect with 10 Gy gamma irradiation in subcutaneous murine tumors which is similar to 160 mg/kg tubulozole administered 6 h before 10 Gy. The enhancing effect is also observed in a clinically relevant radiation dose fractionation schedule whereby eight fractions of 2 Gy each were pretreated 2 h before with 40 mg/kg R 55 104. Further study of this radiochemotherapeutic combination may lead to new clinical applications.

Interaction between the microtubule inhibitor tubulozole and gamma-irradiation in murine tumors in vivo.

The combined effect of the microtubule inhibitor tubulozole and gamma-irradiation has been investigated in vivo in subcutaneous MO4 fibrosarcomas and Lewis Lung carcinomas. A marked interactive effect on tumor growth was observed when 160 mg/kg tubulozole was orally administered before the tumors were treated with 10 Gy radiation. Dose dependency and optimal effect were obtained on tumor growth of MO4 tumor bearing animals when the drug treatment was given 6 hr prior to the irradiation. The optimal pretreatment time coincided with the time at which a peak mitotic index in the tumor tissue was observed. An enhancing effect is also noticed at other doses of radiation in MO4 tumors pretreated 6 hr before with 160 mg/kg tubulozole. The interactive effect is maintained in a clinically relevant dose fractionation schedule whereby 8 fractions of 2 Gy each were pretreated 6 hr before with 80 mg/kg tubulozole. Tubulozole-T, the stereo-isomer of tubulozole, neither exhibits any antimicrotubular action nor exerts an antitumoral effect on its own or in combination with gamma-irradiation. The possible mechanisms of interaction between tubulozole and gamma-irradiation in tumor tissue are discussed.

Dispensing Precision for the SciClone™ Automated Liquid Handling Workstation for 96-Channel Pipetting

One of the first steps in drug discovery involves identification of novel compounds that interfere with therapeutically relevant biological processes. Identification of ‘lead’ compounds in all therapeutic areas included in a drug discovery program requires labor-intensive evaluation of numerous samples in a battery of therapy targeted biological assays. To accelerate the identification of ‘lead’ compounds, Janssen Research Foundation (JRF) has developed in the past an automated high throughput screening (HTS) based on the unattended operation of a custom Zymark tracked robot system. Automation of enzymatic and cellular assays was realized with this system adapted to the handling of microtiter plates. The microtiter plate technology is the basis of our screening. All compounds within our chemical library are stored and distributed in micronic tube racks or microtiter plates for screening. An efficient in-house developed mainframe based laboratory information management system supported all screening activi...

Microtubule Dynamics and the Mitotic Cycle: A Model

In what follows we will give a personal view of how organized microtubule (MT) systems may evolve during the cell cycle. It is based on some of our own experiments and ideas and on those of many other investigators that have devoted attention to these matters. We will not reiterate the argumentation which has led to the various facets of the following hypothesis, which is based on our personal conjecture. This chapter is similar to the concluding remarks of a review in press (9). For a detailed account of the data we refer to this review and Ref. 2–8. Figures 1–4 provide the basic illustrations necessary to follow this short review.