I just like proteases. I used them a lot when I worked at a nucleic acids isolation company called Argylla Technologies. When that company ran out of money, I “borrowed” some leftover Flavourzyme. I was going to make my own special fish proteolytic mix for flavoring soups. I went to the local oriental market and obtained a salmon head. Long story short, I discovered that fish heads contain their own heat and acid activated protease called cathepsin L. Lysosomes are organelles in our cells that digest proteins. If you want to see a salmon head self digest, all you have to do is (1) heat (2) reduce the pH to less than 5 using your favorite vinegar, lime juice, or whatever. Even the bones and eyeballs will self digest. This is an example of something I’d like to set up.
SARS COVID
Corona viruses come with their own self digesting proteases. The SARS corona virus 3CLpro was an area of active research 15 years ago. Cheng-Wen Lin of the Department of Medical Laboratory Science and Biotechnology, China Medical University expressed this protease in E coli. A penta histidine tag was used to purify this viral protease from other material in the bacteria.[1] There are numerous crystal structures of viral 3CLpro proteaseses, all in effort to find protease inhibitors.
Lin and coworkers [1] determined the proteolytic activity o f the SARS‐CoV 3CLpro spectrophotometrically at 440 nm by following the digestion of azocasein (Sigma). 50 mM Tris–HCl, pH 8.5 was used as the buffer at 37 °C. Resulting supernatants centrifuged at 10 000 × g for 10 min, was mixed with 300 μl of 1 N NaOH. The absorbance of the supernatant was measured at 440 nm. This protocol would be easy to replicate in a simple laboratory setting.
Lin and workers [1] used a giant fusion protein constuct containing a linkerto anchor the fusion to a substrate, the 3CLpro protease and a protein S reporter. When this reporter is cleaved off, it can no longer be detected by by an S protein ELISA.
For those in the Copper One Niacin supplement,the cysteine and histidine are potential copper binding sites.
Quercetin, Covid-19 3CLpro
Red wine lovers will appreciate the work of Olga Abian and coworkers at the Instituto Aragonés de Ciencias de la Salud (IACS), 50009 Zaragoza, Spain [2]. These authors also expressed the Coivd-19 3CLpro in E coli. They used a series of biophysical techniques to demonstrate interaction with quecetin [2]. These authors used a self quenching fluorescent peptide probeIn vitro catalytic activity of 3CLpro was determined using a fluorescence resonance energy transfer (FRET) cleavage continuous assay with the peptide substrate (Dabcyl)KTSAVLQSGFRKME(Edans)-NH2 as a substrate. The fluorophore on one end quences the fluorophore on the other end. When the peptide is cleaved in two, the fluorescence increases.
These authors [2] docked used AutoDock Vina software to dock quercetin into PDB structures 6Y2E and 6Y2F. This in silico simulation indicated that Met165 is a key residue that can form both hydrogen bonds and hydrophobic interactions with quercetin. Additional hydrogen bonds can be formed with the two residues Ser144 and Asn142. Charged residues His164 and Glu166 are also predicted to participate in this interaction.
For fans of Copper One Niacin
Jia Gao and coworkers [3] tested the hypothesis that niacin based oral drugs could be repurposed to inhibit the 2CLpro in Covid-19 infections. The beauty of this strategy is that we have already established how this small molecules behave in humans. These authors expressed the Coivd-19 3CLpro in E coli. They measured proteolytic activity using a self quenching fluorescent peptide FITC-AVLQSGFR-Lys(Dnp)-Lys-NH2. This in vitro system was used to screen a library of niacin derived small molecule drugs.
For Copper One niacin customers the interesting thing to note is that the potential copper binding sites in the active site, Post figure 2 are note occupied by niacin. Also note from post figure 1 for the SARS 3CLpro, His41 is required for maximal activity. This explains why niacin is inhibitory.
Many things seem to inhibit this 3CLpro protease. These proteases can be cheaply expressed in bacteria and inhibition of enzymatic activity also assayed for not a lot of money. That so many of these small molecules are already been proven to be safe in humans is an added bonus.
References
- Lin CW, Tsai CH, Tsai FJ, Chen PJ, Lai CC, Wan L, Chiu HH, Lin KH.(2004) Characterization of trans- and cis-cleavage activity of the SARS coronavirus 3CLpro protease: basis for the in vitro screening of anti-SARS drugs. FEBS Lett. 2004 Sep 10;574(1-3):131-7. [PMC free article].
- Abian O, Ortega-Alarcon D, Jimenez-Alesanco A, Ceballos-Laita L, Vega S, Reyburn HT, Rizzuti B, Velazquez-Campoy A.(2020) Structural stability of SARS-CoV-2 3CLpro and identification of quercetin as an inhibitor by experimental screening. Int J Biol Macromol. 2020 Jul 31;164:1693-703. [PMC free article]
- Gao J, Zhang L, Liu X, Li F, Ma R, Zhu Z, Zhang J, Wu J, Shi Y, Pan Y, Ge Y, Ruan K.(2020) Repurposing Low-Molecular-Weight Drugs against the Main Protease of Severe Acute Respiratory Syndrome Coronavirus 2. J Phys Chem Lett. 2020 Sep 3;11(17):7267-7272. [PMC free article]
