Differential Activity of Repurposed Drugs as Receptor Binding Domain Antagonists for Omicron and Native Strains of SarsCov2

Omicron strain is the latest variant of concern of SarsCov2 virus. The mutations in this strain in the S protein Receptor Binding domain (RBD) enable it to be more transmissible as well as escape neutralizing activity by antibodies in response to vaccine. Thus, Omicron specific strategies are need to counter infection by this strain. We investigated a collection of approved drugs shown to antagonize the binding of native strain RBD to human ACE2, for their ability to antagonize binding to Omicron strain RBD. While most of the drugs the drugs that antagonize binding to native RBD are also active for Omicron RBD but some were inactive, namely drugs that contain iodine are completely inactive against Omicron RBD. Our data strongly indicate that presence of a single iodine molecule in the drug renders it inactive against Omicron strain. Thus, there is molecular specificity of drugs for antagonizing Omicron strain RBD versus native strain RBD of this virus. Such information will pave way for specific drugs for Omicron. A pragmatic message from our data is that the often-used iodine containing mouth wash and rises may be ineffective in antagonizing receptor binding of Omicron strain.

Edible formulations of chicken egg derived IgY antibodies neutralize SarsCoV2 Omicron RBD binding to human ACE2

SarsCoV2 virus driven pandemic continues to surge propelled by new mutations such as seen in Omicron strain. Omicron is now rapidly becoming the dominant strain globally with more than 30 mutations in the spike protein. The mutations have resulted in Omicron strain escaping most of the neutralizing antibodies generated by the current set of approved vaccines and diluting the protection offered by the vaccines and therapeutic monoclonal antibodies. This has necessitated the need for newer strategies to prevent this strain from spreading. Towards this unmet need we have developed chicken egg derived anti-RBD IgY antibodies that neutralize the binding of Omicron RBD to human ACE2. Furthermore, we have formulated the edible IgY as flavored beverages to allow for use as oral rinse and prevent the entry of Omicron in the oropharyngeal passage, a major access and accumulation point for this strain in humans.

Preparation of ingestible antibodies to neutralize the binding of SarsCoV2 RBD (receptor binding domain) to human ACE2 Receptor

COVID19 continues to be a serious threat to human health and mortality. There is dire need for new solutions to combat this pandemic especially for those individuals who are not vaccinated or unable to be vaccinated and continue to be exposed to the SARSCoV2. In addition, the emergence of new more transmissible variants such as delta pose additional threat from this virus. To explore another solution for prevention and treatment of COVID 19, we have produced chicken egg derived IgY antibodies against the Receptor binding domain (RBD) of SARSCoV2 spike protein which is involved in binding to human cell ACE2 receptors. The - RBD IgY effectively neutralize the binding of RBD to ACE2 and prevent pseudovirus entry in a PRNT assay. Importantly our anti-RBD IgY also neutralize the binding of Sars CoV2 delta variant RBD to ACE2. Given that chicken egg derived IgY are safe and permissible for human consumption, we plan to develop these ingestible antibodies for prevention of viral entry in the oropharyngeal and digestive tract in humans as passive immunotherapy.

Quercetin-3-O-rhamnoside from Euphorbia hirta protects against snake Venom induced toxicity.

BACKGROUND: The plant Euphorbia hirta is widely used against snake envenomations in rural areas and it was proved to be effective in animal models. Therefore, the scientific validation of its phytoconstituents for their antiophidian activity is aimed in the present study. METHODS: E. hirta extract was subjected to bioactivity guided fractionation and the fractions that inhibited different enzyme activities of Naja naja venom in vitro was structurally characterized using UV, FT-IR, LC-MS and NMR spectroscopy. Edema, hemorrhage and lethality inhibition activity of the compound were studied in mice model. In addition, molecular docking and molecular dynamic simulations were also performed in silico. RESULTS: The bioactive fraction was identified as Quercetin-3-O-α-rhamnoside (QR, 448.38 Da). In vitro experiments indicated that protease, phospholipase-A(2), hemolytic activity and hemorrhage inducing activity of the venom were inhibited completely at a ratio of 1:20 (venom: QR) w/w. At the same concentration, the edema ratio was drastically reduced from 187% to 107%. Significant inhibition (93%) of hyaluronidase activity was also observed at a slightly higher concentration of QR (1:50). Further, in in vivo analysis, QR significantly prolonged the survival time of mice injected with snake venom. CONCLUSION: For the first time Quercetin-3-O-α-rhamnoside, isolated from E. hirta, has been shown to exhibit anti-snake venom activity against Naja naja venom induced toxicity. GENERAL SIGNIFICANCE: Exploring such multifunctional lead molecules with anti-venom activity would help in developing complementary medicine for snakebite treatments especially in rural areas where anti-snake venom is not readily available.

Protective effect of Euphorbia hirta and its components against snake venom induced lethality.

ETHNOPHARMACOLOGICAL RELEVANCE: Despite the use of snake anti-venom therapy, herbal medicine is still in practice to treat snakebites. Euphorbia hirta is traditionally used as antidote for snakebites and also for numerous other ailments. However, the scientific evidence for its anti-snake venom property is still lacking. MATERIALS AND METHODS: Methanolic extract of E. hirta was evaluated for anti-venom activity under in vitro and ex vivo conditions. Histopathological changes in the vital organs of the mice were also monitored. UHPLC-SRM/MS was used to estimate the phenolic constituents whereas GC-MS analysis was performed to analyze the volatile metabolites present. The major compound was further evaluated for its contribution to the overall inhibitory potential of the extract. RESULTS: Methanolic extract of E. hirta completely inhibited the venom enzymes under in vitro and reduced the edema ratio. The extract increased the survival time (>24h) of mice which was further evidenced by histopathological analysis of vital organs. Phytochemical analysis revealed higher content of phenolic (144 mg/g extract) compounds in the extract. UHPLC-SRM/MS demonstrated that ellagic acid, gallic acid and quinic acid are the major phenolics whereas GC-MS analysis revealed pyrogallol as the major constituent (60.07%) among the volatile components of the extract. It was also shown that pyrogallol has the ability to differentially inhibit venom protease but not phospholipase A2. CONCLUSION: The present study confirmed that E. hirta methanolic extract was able to completely inhibit Naja naja venom induced toxicity under in vitro as well as ex vivo conditions, thus providing scientific evidence to its traditional use.

Formulation and characterisation of antibody-conjugated soy protein nanoparticles--implications for neutralisation of snake venom with improved efficiency.

The present study reports the formulation of soy protein nanoparticles and its conjugation to antivenom. The conditions for nanoparticle formation were optimised by considering particle size, count rate, stability and zeta potential. The smallest particle size of 70.9 ± 0.9 nm with a zeta potential of -28.0 ± 1.4 mV was obtained at pH 6.2, with NaOH 5.4 % and 28 µg/mg glutaraldehyde. The nanoparticle was conjugated with antisnake venom immunoglobulins (F(ab')2 fragments) using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide. TEM analysis indicated the increased size of particle to 600 nm after conjugation to antivenom. Further, in vitro studies indicated that conjugated antibodies inhibited the activity of protease, phospholipase and hyaluronidase enzymes of Bungarus caeruleus venom more efficiently than the free antivenom. This is the first report on the use of protein nanoparticles for conjugating snake venom antibodies and their implications for neutralising snake venom enzymes with increased efficiency.

Inhibition of Naja naja venom enzymes by the methanolic extract of Leucas aspera and its chemical profile by GC-MS.

PURPOSE: The present investigation was aimed at evaluating the anti-ophidian properties of ethnomedicinal herb Leucas aspera against Indian cobra, Naja naja venom enzymes. METHODS: Methanolic extract of Leucas aspera was evaluated, in vitro, for its ability to inhibit the major enzyme activities of Naja naja venom including protease, phospholipase A2, hyaluronidase and hemolytic factors. The type of phytochemicals present in the extract was analyzed. Also, the major phytoconstituents in the extract was determined by gas chromatography-mass spectrometry (GC-MS). RESULTS: Venom protease and hyaluronidase activities (two isoforms) were completely (100%) neutralized by the L. aspera methanolic extract at ratio of 1:50 w/w (venom: plant extract) and venom hemolytic activity was also completely neutralized at a ratio of 1:80 w/w by the plant extract. However, the extract failed to neutralize phospholipase A2 activity even at the highest concentration used. Phytochemical analysis revealed the presence of alkaloids, acidic compounds, flavonoids, steroids and cardiac glycosides in the extract. GC-MS analysis indicated that a total of 14 compounds were present in the extract. The major bioactive constituents were found to be 6-octadecenoic acid (32.47%), n-hexadecanoic acid (25.97%), and 17-octadecen-14-yn-1-ol (14.22%) along with the minor constituents, sitosterol (2.45%) and stigmasterol (2%), which was previously reported to exhibit antivenom activity. CONCLUSION: The results obtained demonstrate for the first time that the methanolic extract of Leucas aspera possesses anti-venom activity and could be considered as a potential source for the anti-ophidian metabolites.