Enhancement of the antiviral activity of pyrimidine derivatives against mengovirus by visible light.

Eleven pyrimido-pyrimidine derivatives, seven with significant antiviral activity against Mengovirus, five against Coxsackie B1 virus and four antiviral negative compounds were tested for their photosensitizing ability. All seven compounds with antiviral activity in vitro showed an enhanced antiviral action against Mengovirus under irradiation with visible light, a fact that may be caused by photodynamic processes. It was tried to correlate the oxidation potentials of sensitizers with their photodynamic activity. By means of mass-spectrometric investigations, molecular fragmentation was examined following thin layer chromatography (TCL) before and after irradiation. Furthermore, binding affinity to biopolymers (BSA and RNA) was investigated to reveal conformity in differences of antiviral activity. The main results are the following: 1. Generally, strong antiviral activity can be correlated with strong binding affinity. 2. No significant correlation could be detected between oxidation potentials of antiviral compounds and their enhanced antiviral activity under irradiation conditions, although in some cases sensitizer with higher oxidation potentials are more effective than those with lower ones. 3. The lower the photostability of the compounds the higher was the light-induced antiviral activity. 4. No alteration of the molecular ion peak and fragmentation pattern before and after irradiation was indicated by means of mass-spectrometry and TLC using fairly comparable conditions.

Antiviral pyrimidine derivatives: binding with biopolymers and photosensitization.

Two antiviral pyrimidine derivatives, known to partially inhibit RNA dependent RNA polymerase of Mengo virus in a cell-free system, were found to bind with biopolymers. The antiviral compounds, at concentration of 100 and 50 muM and lower, showed complete inhibition of the virus-induced cytopathic effect and plaque reduction. Both compounds showed fluorescence emission with a maximum at about 500 nm under the influence of UV light of the wavelength of 366 nm. The fluorescence intensity increased upon addition of aqueous solutions of DNA, RNA and human serum albumin. This indicates that both compounds are capable of binding with nucleic acids and proteins. Based on the binding experiments alone it cannot be decided whether interference with nucleic acid template activity, enzyme function, or both are responsible for the virostatic action. Irradiation with violet light considerably enhances the virostatic activity, a fact that may be caused by photodynamic processes.

Effect of ultraviolet light on mengovirus: formation of uracil dimers, instability and degradation of capsid, and covalent linkage of protein to viral RNA.

UV irradiation of purified mengovirus resulted in a very rapid inactivation of the infectivity of the virions (D(37) [37% survival dose] = 700 ergs/mm(2)) which correlated in time with the formation of uracil dimers in the viral RNA. During the first 2 min of irradiation, an average of 1.7 uracil dimers were formed per PFU of virus inactivated. Hemagglutination activity of the virions began to decrease only after a lag period of about 5 min and at a much lower rate (D(37) = 84,000 ergs/mm(2)). This decrease coincided in time with the appearance of altered proteins in the capsid and a structural change in the capsid. Although 10- to 20-min irradiated virions appeared intact in the electron microscope and sedimented at 150S in sucrose density gradients, the RNA of the virions became accessible to RNase and extractable by low concentrations of sodium dodecyl sulfate, and the virions broke down upon equilibrium centrifugation in CsCl gradients. During longer periods of irradiation (30 to 60 min), a progressively greater proportion of the virions were converted to 14S protein particles and 80S ribonucleoprotein particles composed of intact viral RNA and about 30% of the capsid proteins, alpha, beta, and gamma. Empty capsids were not detectable at any time during 60 min of irradiation, by which time disruption of the virions was complete. Irradiation of complete virions also resulted in an increased sedimentation rate of the viral RNA and in the covalent linkage to the viral RNA of about 1% of the total capsid protein in the form of heterogeneous low-molecular-weight polypeptides. The two observations seem to be causally related, since irradiation of isolated viral RNA did not result in an increase in sedimentation rate of the RNA, even though uracil dimer formation in viral RNA occurred at about the same rate and to the same extent whether intact virions or viral RNA were irradiated.