A simple and rapid pipeline for identification of receptor-binding sites on the surface proteins of pathogens.

Ligand-receptor interactions play a crucial role in the plethora of biological processes. Several methods have been established to reveal ligand-receptor interface, however, the majority of methods are time-consuming, laborious and expensive. Here we present a straightforward and simple pipeline to identify putative receptor-binding sites on the pathogen ligands. Two model ligands (bait proteins), domain III of protein E of West Nile virus and NadA of Neisseria meningitidis, were incubated with the proteins of human brain microvascular endothelial cells immobilized on nitrocellulose or PVDF membrane, the complex was trypsinized on-membrane, bound peptides of the bait proteins were recovered and detected on MALDI-TOF. Two peptides of DIII (~916 Da and ~2003 Da) and four peptides of NadA (~1453 Da, ~1810 Da, ~2051 Da and ~2433 Da) were identified as plausible receptor-binders. Further, binding of the identified peptides to the proteins of endothelial cells was corroborated using biotinylated synthetic analogues in ELISA and immunocytochemistry. Experimental pipeline presented here can be upscaled easily to map receptor-binding sites on several ligands simultaneously. The approach is rapid, cost-effective and less laborious. The proposed experimental pipeline could be a simpler alternative or complementary method to the existing techniques used to reveal amino-acids involved in the ligand-receptor interface.

Anti-HIV, anti-poxvirus, and anti-SARS activity of a nontoxic, acidic plant extract from the Trifollium species Secomet-V/anti-vac suggests that it contains a novel broad-spectrum antiviral.

Enveloped animal viruses such as human immunodeficiency virus (HIV), hepatitis B virus, hepatitis C virus, human papillomavirus, Marburg, and influenza are major public health concerns around the world. The prohibitive cost of antiretroviral (ARV) drugs for most HIV-infected patients in sub-Saharan Africa and the serious side effects in those who have access to ARV drugs make a compelling case for the study of complementary and alternative therapies. Such therapies should have scientifically proved antiviral activity and minimal toxic effects. A plant extract, Secomet-V, with an anecdotal indication in humans for promise as an anti-HIV treatment, was investigated. Using a previously described attenuated vaccinia virus vGK5, we established the antiviral activity of Secomet-V. Chemical analysis showed that it has an acidic pH, nontoxic traces of iron (<10 ppm), and almost undetectable levels of arsenic (<1.0 ppm). The color varies from colorless to pale yellow to dark brown. The active agent is heat stable at least up to sterilizing temperature of 121 degrees C. The crude plant extract is a mixture of several small molecules separable by high-pressure liquid chromatography. The HIV viral loads were significantly reduced over several months in a few patients monitored after treatment with Secomet-V. Secomet-V was also found to have antiviral activity against the SARS virus but not against the West Nile virus. Secomet-V, therefore, is a broad-spectrum antiviral, which possibly works by neutralizing viral infectivity, resulting in the prevention of viral attachment.

Herbal complement inhibitors in the treatment of neuroinflammation: future strategy for neuroprotection.

The upregulated complement system plays a damaging role in disorders of the central nervous system (CNS). The classical and alternate pathways are two major pathways activated in neuroinflammatory disorders such as Alzheimer's disease, multiple sclerosis, traumatic brain injury, spinal cord injury, HIV-associated dementia, Parkinson's disease, and mad cow disease. Failure of currently available anti-inflammatory agents, especially cyclooxygenase inhibitors, in offering significant neuroprotection in large epidemiologic clinical trials of CNS disorders suggests an urgent need for the development of new neuroprotective agents. The positive preclinical outcomes in treating CNS disorders by complement regulatory molecules, such as vaccinia virus complement control protein, suggest the possibility of using complement-inhibitory molecules as neuroprotective agents. Several active ingredients of herbal origin are found to have complement-inhibitory activity. These herbal ingredients along with other anti-inflammatory roles might be useful in treating neuroinflammation associated with CNS disorders. Active ingredients of herbal origin with complement inhibitory ingredients are summarized and classified according to their chemical nature and specificity towards the major pathways activating the complement system. The structure activity relationship of some specific examples is also discussed in this report. This information might be helpful in formulating a natural panacea against complement-mediated neuroinflammation.