ABSTRACT
BACKGROUND: Host proteases have been shown to play important roles in many viral activities such as entry, uncoating, viral protein production and disease induction. Therefore, these cellular proteases are putative targets for the development of antivirals that inhibit their activity. Host proteases have been described to play essential roles in Ebola, HCV, HIV and influenza, such that specific protease inhibitors are able to reduce infection. RSV utilizes a host protease in its replication cycle but its potential as antiviral target is unknown. Therefore, we evaluated the effect of protease inhibitors on RSV infection. METHODS: To measure the sensitivity of RSV infection to protease inhibitors, cells were infected with RSV and incubated for 18 h in the presence or absence of the inhibitors. Cells were fixed, stained and studied using fluorescence microscopy. RESULTS: Several protease inhibitors, representing different classes of proteases (AEBSF, Pepstatin A, E-64, TPCK, PMSF and aprotinin), were tested for inhibitory effects on an RSV A2 infection of HEp-2 cells. Different treatment durations, ranging from 1 h prior to inoculation and continuing for 18 h during the assay, were evaluated. Of all the inhibitors tested, AEBSF and TPCK significantly decreased RSV infection. To ascertain that the observed effect of AEBSF was not a specific feature related to HEp-2 cells, A549 and BEAS-2B cells were also used. Similar to HEp-2, an almost complete block in the number of RSV infected cells after 18 h of incubation was observed and the effect was dose-dependent. To gain insight into the mechanism of this inhibition, AEBSF treatment was applied during different phases of an infection cycle (pre-, peri- and post-inoculation treatment). The results from these experiments indicate that AEBSF is mainly active during the early entry phase of RSV. The inhibitory effect was also observed with other RSV isolates A1998/3-2 and A2000/3-4, suggesting that this is a general feature of RSV. CONCLUSION: RSV infection can be inhibited by broad serine protease inhibitors, AEBSF and TPCK. We confirmed that AEBSF inhibition is independent of the cell line used or RSV strain. The time point at which treatment with the inhibitor was most potent, was found to coincide with the expected moment of entry of the virion with the host cell.
Subject(s)
Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus, Human/drug effects , Respiratory Syncytial Virus, Human/pathogenicity , Serine Proteinase Inhibitors/pharmacology , Sulfones/antagonists & inhibitors , Virus Internalization/drug effects , A549 Cells , Aprotinin/pharmacology , Cell Line , Cell Survival/drug effects , Endopeptidases/drug effects , Humans , Kinetics , Leucine/analogs & derivatives , Leucine/antagonists & inhibitors , Pepstatins/antagonists & inhibitors , Time Factors , Tosylphenylalanyl Chloromethyl Ketone/antagonists & inhibitors , Viral Proteins/metabolismABSTRACT
Gallic acid, one of the most abundant plant phenolic acids, has been modified to cathepsin D protease inhibitors. The strategy of modification was proposed basing on some previously reported structure and activity relationship (SAR) studies. The synthesized naphthophenone fatty acid amide derivatives have been evaluated for in vitro cathepsin D inhibition activity. Two of them have shown significant inhibition activity with IC(50) values of 0.06 and 0.14 microM, respectively, as compared against pepstatin (0.0023 microM), the most potent inhibitor known so far. The study revealed that such attempts on gallic acid based pharmacophores might result in potent inhibitors of cathepsin D.
Subject(s)
Amides/chemistry , Cathepsin D/antagonists & inhibitors , Cathepsin D/metabolism , Fatty Acids/chemistry , Gallic Acid/chemical synthesis , Gallic Acid/pharmacology , Ketones/chemistry , Naphthalenes/chemistry , Gallic Acid/chemistry , Molecular Structure , Pepstatins/antagonists & inhibitors , Pepstatins/metabolism , Structure-Activity RelationshipABSTRACT
In a series of experiments dealing with the effects of angiotensin I (AI) and angiotensinogen on isolated rat aorta we observed that pepstatin A was able to induce contractile effects by itself. The endothelin pathway was excluded by the inhibitory effects of captopril, chymostatin and amastatin. In addition, few preliminary experiments showed that the contractile effects of pepstatin A were inhibited by the pretreatment with losartan, an antagonist of AT1 angiotensin receptors. Pepstatin A-induced contractile effects on isolated rat aorta were inhibited with high potency by captopril, chymostatin and amastatin and were totally blocked by captopril + amastatin and captopril + chymostatin. Finally, we concluded that the pepstatin A-induced contractile effects on isolated rat aorta rings are dependent on an angiotensinogen vascular pool, compulsory involve an angiotensin-converting enzyme-1 (ACE-1) mediated pathway and one or more non-classical pathways for the generation of angiotensin peptides. Further experiments are necessary to elucidate the mechanisms associated to pepstatin A-induced effects.
Subject(s)
Aorta/drug effects , Muscle, Smooth, Vascular/drug effects , Pepstatins/pharmacology , Protease Inhibitors/pharmacology , Angiotensin I/physiology , Angiotensinogen/physiology , Animals , Aorta/physiology , Male , Muscle, Smooth, Vascular/physiology , Pepstatins/antagonists & inhibitors , Rats , Rats, WistarABSTRACT
It was found that the human and bovine vitreous contains a proteolytic enzyme(s) which demonstrates characteristic features of cathepsin D. It acts in acidic pH with various activity, dependent on the condition of the eye and actively digests native and denatured protein substrates. It demonstrates a high susceptibility to the inhibitory action of pepstatin. The role of this enzyme in physiology and pathology of the eye is discussed.
