Thursday, June 4, 2020

Tu YouYou's speech at the 2002 China-Africa Forum on Traditional Medicine

There is a famous person who really should be a lot more famous, even after having earned the Nobel Prize in Physiology or Medicine in 2015. Her name is Tu YouYou.  She discovered artemisinin, which is now the first line malaria medication for infected cases.  What's even more interesting and perhaps more deserving of fame (in light of the result of course) was that she did so by mining traditional medicine systems, in this case Traditional Chinese Medicine, to find this therapeutic.  This is one of those medications that are probably most easily and appropriately termed a 'cure', although the medical profession looks down at that term for anything because of the expectations it instills in the patient and their loved ones (and there's always an exception, complication, or side effect in some small number of patients relative to the total number of treated).

Anyway I was at the KEnya Medical Research Institute hq the other day, in their Traditional Medicine Research department.  There was a binder there from 2002 with the agenda and speeches from an Africa-China traditional medicine research convocation in Beijing.  There was a speech of hers inside, and I was given permission to transcribe it.  Here it is:

"

China-Africa Forum on Traditional Medicine convened from 20-22 October in Beijing

Qinghaosu [ed. note: Artemisinin] - the Crystal of Traditional Anti-malaria Chinese Medicine

Tu Youyou
Director, Research Center for Qinghao and Qinghaosu, China Academy of Traditional Chinese Medicine, Beijing, 100700, China

I am a researcher from the China Academy of traditional Chinese medicine.  Traditional Chinese medicine (TCM) with its unique theory is shaped from the long history of medical practice and experiences accumulation of the Chinese nation.  TCM protects the development and multiplication of the nation.  THe flourishing of TCM today is based on the continuous invention nad innovation in its history.  In 11th century BC, anatomic terms of human body and descriptions of a number of diseases were recorded by inscriptions on bones and tortoise shells.  The Yellow Emperor's Internal Classic (Qin dynasty and Han dynasty) and Treatise on Diseases (by Zhang Zhongjing, East Han dynasty) establish the theoretical basis of TCM, and guided the clinical practice of the medicine.  Medicine and drugs are inseparable.  Chinese herbal medicine has a long history as the one of TCM.  Over a hundred of herbal medicines and treatment and/or prevention for dozens of diseases are registered in Canon of mountains and Sea (3rd century BC). Historical Record carries the following message " Shen Nong (the god of agriculture) tasted hundreds of herbs and initiated the medicine".  Shen Nong is certainly a legend character.  However, Shen Nong's Herbal Classic is a reality.  It carries 365 kinds medicines from herbal, animal and mineral origins.  The newly-revised Materia Medica (659 AD) was implemented in Tang dynasty. In 1578 , the well-known Compendium of Materia Medica was completed by Li Shizhen of Ming dynasty, and printed the cut blocks in 1593. After founding of new China, the Academy of Traditional Chinese Medicine, affiliated to the Ministry of Health, was set up in 1955. In 1983, seven WHO collaborating centers for TCM in China were established. The Institute of Chinese Materia Medica, China Academy of TCM is one of them.

In 1960's, malaria was epidemic when Vietnam was in the flames of war. It has been an urgent need for new antimalarial drugs due to the problem of chloroquine-resistant strains of P. Facliparum. Globally, hundreds of millions of people were annually suffered from the disease, and the mortality was increased sharply. A large scale screening for antimalarial drugs was carried out home and abroad at that time, without satisfactory results. Our project for developing new antimalarial drugs was set up under the above mentioned situation in 1969. Malaria is a common disease for the world, and recorded in a medical literature (12 century, BC) in China.  I graduated from the Department of Pharmacy, Beijing Medical College, and systematically studied TCM after employed.  I firmly believe that TCM with this long history is a great treasure-house, and efforts should be made to explore its essence. On the basis of collection and analysis of traditional prescriptions, over two hundreds of herbs and three hundreds and eighty abstracts from them were screened by malarial models of mouse or monkey.  The screening showed positive results in October, 1971. We isolated and purified the effective compound from the traditional Chinese herb, Artemisia annua L, in 1972. It was named Qinghaosu after the herb's pronunciation in Chinese. Qinghaosu showed 100% inhibition to malaria parasites for monkey and mouse.  In 1973, clinical trials verified the efficacy of Qinghaosu. A new antimalarial drug was finally appeared. Qinghaosu is a compound of sesquiterpene lactone with a peroxide. It is just what we were looking for - the antimalarial drug with totally new chemical structure. The antimalarial effect of Artemisia annua L was firstly described in "The handbook of prescriptions for emergencies" compiled by Ge Hong (281-340 AD), the physician and alchemist in East Jin dynasty. The invention of Qinghaosu is benefited from TCM. It is a good illustration of the treasure and essence in TCM and the potential for contribution to the world medicine. Qinghaosu is a drug with high efficacy, fast-action and safe. In October of 1981, I delivered a talk on the chemical research of Qinghaosu during the international symposium of Qinghaosu held in Beijing. The talk evoked great attention. Qinghaosu was appreciated as "a new antimalarial drug with importance of pointing direction for drug synthesis and design". New drug certificate for Qinghaosu was issued as the first category drug in 1986, and the one for dihydro-Qinghaosu later on. Dihydro-Qinghaosu is a derivative of Qinghaosu with two hydroxides. It provides basis for further development of derivatives of Qinghaosu. The achievements gained the National Invention Award and the Top Ten National Award for Progress in Science and Technology accordingly.

For our good wishes, the 21st century should be a better one with magnificent prospects. Although we can roam in the heavens, and the world is connected closely by the internet, but we still have to face the cruel facts of local wars and life-threatening diseases.  Malaria is still a dangerous enemy to mankind in the new century. An airplane crash may kill hundreds of people, and results in an impact of shake and grief for the living people for a long time to come.  However, we sometimes neglect the significant tragedy. Malaria, as the tropical disease, is cruelly killing the poor people in the developing countries. Some 500 million infections are globally reported, and some 3,000 children are killed daily in Africa only.  The simple figures tell us that ten abuses [editors note: Airbuses probably] full of children were crashed every day. What a nightmare to the mankind! We get to fight back. "Roll back malaria" is the fighting target for the new century setup by WHO. I believe Qinghaosu and its derivatives are the right weapons for the combat. We sincerely hope that the invention of Qinghaosu will save thousands of endangered people from the verge of death daily. Research Center for Qinghao and Qinghaosu was established at the China Academy of TCM due to the invention of Qinghaosu. The center carries out systematic researches on Qinghaosu, and gains new progresses in compound prescriptions with better therapeutic efficacy ( increased 7.6 folds ) and in new applications of Qinghaosu.  We are looking forward to cooperating with our African colleagues. I heartily hope that Qinghaosu will contribute more for the benefit of human health in the 21st century and for the bright future of mankind.

"
















Thursday, April 30, 2020

45 Flavonoids vs. SARS-CoV's 3CLpro under Autodock Vina


In Silico effect of Flavonoids on SARS-CoV 3CLpro progressively targeting A and B-chains' HIS41-CYS145 interfaces


Results:


Micro-discussion:
So while I've been a very strong proponent of Hesperidin based on in vitro results. benign cytox values, inherent known safety, and wide suppy availability, this Autodock Vina docking study shows stronger hits in 1. Hinokiflavone, 2. Luteolin-O-7-rutinoside, 3. Eriocitrin, 4. Sulcatone A, and 5. Amentoflavone.
As amentoflavone is the only one of the above with known outperforming in-vitro efficacy demonstration on SARS-CoV 3CLpro, while showing two orders of magnitude margin for cytotoxicity on Vero cells, I advocate that in vitro trials targeting phytotherapies focus on amentoflavone as applied to (e.g. e.coli-expressed) SARS-CoV-2 3CLpro proteins.

Source data:
https://docs.google.com/spreadsheets/u/1/d/19RGHp9ls3fdjDPYgT-YtNwWXsMwI9f3Glqz6KnedK0M/edit?usp=drive_web&ouid=117587353864850994495

PDB File: 2duc.pdb

Ligand Files: PubChem 3D Conformers

Citations:
Yu Wai Chen, Chin-Pang Bennu Yiu, Kwok-Yin Wong, (Hong Kong Polytechnic University)
Prediction of the SARS-CoV-2 (2019-nCoV) 3C-like protease (3CLpro) structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidate”, F1000Research, 2020

O. Trott, A. J. Olson,                                        
AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading, Journal of Computational Chemistry 31 (2010) 455-461 DOI 10.1002/jcc.21334  

Lin, Tsai, Tsai et al. (China Medical University)
Anti-SARS coronavirus 3C-like protease effects of Isatis indigotica root and plant-derived phenolic compounds", Antiviral Research, Volume 68, Issue 1, October 2005, Pages 36-42.


Ryu, YB et al. (Korea Research Institute of Bioscience and Biotechnology)
"Biflavonoids from Torreya nucifera displaying SARS-CoV 3CL(pro) inhibition.", Bioorg Med Chem., 2010 Nov 15; 18(22):7940-7.
DOI 10.1016/j.bmc.2010.09.035

Michel F. Sanner.
Python: A Programming Language for Software Integration and Development. J. Mol. Graphics Mod., 1999, Vol 17, February. pp57-61

Morris, G. M., Huey, R., Lindstrom, W., Sanner, M. F., Belew, R. K., Goodsell, D. S. and Olson, A. J. (2009) Autodock4 and AutoDockTools4: automated docking with selective receptor flexiblity. J. Computational Chemistry 2009, 16: 2785-91

Wednesday, April 29, 2020

SARS-CoV-2 3CLpro- Flavonoids - (Autodock 4)

So I've been doing a lot of flavonoid in silico docking runs on SARS-CoV-2's 3CLpro (pdb file 2duc) protein. Posting results here for posterity.

Image

Monday, April 13, 2020

One instance of research papers permalink list

(for storing, not for reading)

https://drive.google.com/file/d/1Rs9gNn_VTOZBtO5Ffmg5veTUR34PaO9u/view?usp=sharing
https://drive.google.com/file/d/1fVADH8AmlOLTkP7GkTcMTXMH8QYFRsvq/view?usp=sharing
https://drive.google.com/file/d/1uY2PICUp0RdNvwM4k_FwNuVw2hXyOCIQ/view?usp=sharing
https://drive.google.com/file/d/1mHITkOY22LYs2d89_vyRBeAqOOF6a0rQ/view?usp=sharing
https://drive.google.com/file/d/1mPD8o4JVDCLAu-P8eiObxboOyiSJrsvb/view?usp=sharing
https://drive.google.com/file/d/1tQVb8a-r-gEs8yFpO2bdJ6KalRn87ykj/view?usp=sharing

Saturday, March 28, 2020

I failed my first Intro Chemistry course, only barely passing my second. And this is my literature review on coronavirus.

For the tl;dr, scroll to the bottom.

- - - - - -

So with the coronavirus pandemic going on, like everyone else I've been stuck home with little more to do outside of work than to read up on this beast with the minor privilege of being able to ask friends who are more competent in such matters than me about it.  When I see friends and family in my hometown of San Francisco who work in ER wards and nearby county clinics posting their stories and PPE fundraisers on Facebook, I know that something is very wrong, and so in spite of my lay credentials ( I failed chemistry my first semester in university, only to pass within an inch of my life the 2nd semester), feel compelled to post what I've learned about it.

I'm going to describe by way of outlining my learning progression, because I want it to be clear how I got from A to B to C.

As the epidemic in China was starting to wane, but picking up elsewhere, my anxiety grew.  Seeing reports that an HIV antiviral Kaletra (lopinivir and ritonavir) was showing early anecdotal success in patients in China, I asked my Chinese b-school classmates for links to scholarly papers gaining currency on the topic in China, simply so that I could try to reduce my anxiety. 

One of the papers they identified was this, a preprint from Hong Kong Polytechnic University cleverly using Python tools and a drug database to do a drug-docking study:

In-silico drug screening against SARS-nCoV-2:
https://f1000research.com/articles/9-129

I remembered from a UCSF biotech researcher I roomed with two decades ago that proteins are capable of interacting if they have a 3D structural match and complementary chemical bonds at their active sites - like two puzzle pieces fitting together -  so I appreciated the approach and marveled how far the technology had come in 20 years that anyone with Python could do such simulations.  These simulations are called 'in silico', to distinguish themselves from 'in vitro' (testing a compound on actual animal cells, typically in a petri dish), and 'in vivo', (testing the efficacy of a compound in a live animal or even a live human population).

Reading that paper sensitized me to the proteins that are being targeted by drug designers during this epidemic.  For example, there is the protein that makes the virus's spikes. There are other proteins required for the virus to enter a cell and begin replicating itself using the cell's machinery.  Altogether there are some 20-30 unique proteins that make up this coronavirus, which is technically called SARS-CoV-2.  (Side note: A lot of papers out of China were calling the virus 2019-nCoV, so make sure to use that keyword if you ever want to research papers out of China especially from the early period of the pandemic).

The protein the researchers from Hong Kong Polytechnical University were targeting is called 3CLpro.  They usefully compare it to the coronavirus from the 2003 outbreak:

" All 11 3CLpro sites2 are highly conserved or identical (Extended data7, Table S1), inferring that their respective proteases have very similar specificities. The 3CLpro sequence of SARS-CoV-2 has only 12 out of 306 residues different from that of SARS-CoV (identity = 96%)."

They map the 'in silico' efficacy of FDA-approved prescription drugs, and they also study other non-prescription compounds that are typically plant-derived.  They use the binding energy as the figure of merit for the efficacy of a drug or compound to affect the 3CLpro protein.  The logic goes, if you can bind most strongly to one of the virus' protein sites, then you're probably inhibiting the virus' performance somewhat.

Since I could see there was already a lot of scholarly work happening on the approved presription antivirals, such as lopinivir/ritonavir and remdesivir, I began searching on 3CLpro with regards to the non-prescription compounds that ranked quite highly in the HK Polytechnic U researchers' study -specifically hesperidin and diosmin.  Hesperidin and diosmin are found prevalently in orange peels and orange juice, and hesperidin is also found prevalently in peppermint.

On performing the search, one of the earliest hits was this:

https://www.sciencedirect.com/science/article/pii/S0166354205001257
which is a 2005 paper out of China Medical University in Taiwan covering in-vitro drug testing on SARS-nCoV using Vero cells.

This 2005 paper performed in the wake of the original SARS epidemic describes the compounds they tested against the 3CLpro protein in a type of standard animal cell test called "Vero" cells. (These are cells that come from African Green Monkeys, and have been used since the 1950's as a standard cell test that researchers could easily replicate findings against)

The 2005 Taiwan paper also tests compounds that the HKPU study didn't even look at, like Indigo and Sinigrin.  Evidently they didn't even have the benefit of in silico screenings, as their motivation for testing these choices of compounds was based on:

"Isatis indigotica root and phenolic Chinese herbs were frequently used for the prevention of SARS during the SARS outbreaks in China, Hong Kong, and Taiwan"  and "In addition, several herb-derived phenolics aloeemodin, hesperetin, quercetin, and naringenin have been accredited with antiviral effects against poliovirus, vesicular stomatitis virus, Sindbis virus, herpes simplex virus types 1 and 2, parainfluenza virus, and vaccinia virus" (with trailing citations)

As the authors acknowledge, it turns out that hespiritin performs well in inhibiting 3CLpro's cleaving action, which is otherwise necessary for the virus to propagate, at relatively low concentrations compared with the other compounds, at 8 micrograms per mL.

Finally, and usefully, the Taiwanese researchers looked at what upper limit of quantity of the compounds under test would actually kill the test cells.  Lots of compounds kill viruses in a petri dish, but you want to know which of those compounds doesn't also kill the cells they are intended to protect.  As they say after all, "the dose makes the poison."

So hespiritin starts to look interesting, but so does sinigrin, because ostensibly it is so benign to the test cells that it is beyond the top of their scale in terms of ability to harm the cells.  Sinigrin is common in many green vegetables such as brussels sprouts and collard greens.  It's also what gives horseradish its unique test.

Also at this point, note the slightly different spelling hesperidin -> hesperitin.  This will become relevant later, but essentially hesperidin is the naturally-occurring compound, and hesperitin is what the human body metabolizes hesperidin into.

So I started to think about possible administration routes of these compounds.  The two that stand out are 1) ingestion, because it's the easiest and most popular form of taking medication, and 2) (since this manifests as a respiratory and pulmonary infection . . . inhalation.

But then I felt stuck.  Things are looking up, but I don't know what to do next, or even if my approach to reviewing the research is valid.  On the advice of a friend who has a PhD in Systems Biology and practicing in the pharmaceutical industry, he suggested I look up the safety of these administration methods with regards to these compounds.

After a lot of cited online research later, I learned that Sinigrin all-too-easily becomes poisonous at high ingestion (and inhalation (!) ) doses!  Anyone who's ever taken a deep whiff of horseradish or sushi wasabi can speak to this.  So this literature review helpfully enabled ruling it out, leaving Hesperidin as the only remaining candidate to show high in vitro efficacy at accessibly low concentrations.

Then we naturally want to learn how can this compound be had in therapeutically effective doses?  For this I did a bit of math based on the Taiwan paper.  The Taiwan researchers identify the effective concentration for hesperidine to be 2.5 micrograms (mcg or μg) per mL (presumably aqueous).  Taking simple orange juice as the most available vessel for this, and treating the human body as having the same density as water (1 kg per litre) and assuming uniform concentration of the ingested compound in the body, then a 100-kg person, (say a heavier male) would need 250000 μg, or 250 mg, of hesperidine maintained over some period relevant to the virus' generation cycle.

According to this really helpful database of concentrations of various compounds in foods, we find that production orange juice contains 26 milligrams / 100ml of hesperidin.  Hesperitin is about half the molar weight of hesperidin, so we need to treat this as an effective 13 milligrams / 100ml of hesperitin.  So how much OJ does a 100-kg person have to retain without excreting any through urine, in order to reach the dose level indicated by the Taiwan study?  The simple math answer is 1.9 litres.  The average human's weight, 62-kg, would need about 1.2 litres.

While there are many questions remaining that, absent further expert advice, could affect the efficacy of orange juice on either prophylactically protecting against a coronavirus infection, or tamping down a nascent existing infection, considering the universally-accepted safety of orange juice consumption in the world, the choice to drink orange juice is essentially a zero-cost option.  It doesn't pose a health-risk anywhere close to these quantities (the equivalent of having passed phase-1 clinical trials for pharmaceutical drugs), its cheap for developed country consumers, and its widely available, and in all likelihood is sitting inside your fridge as I write.

Don't like drinking all that orange juice?  Well hesperidine (paired with diosmin, which is anyway also naturally prevalent in the same orange juice) is manufactured in pill form, with the hesperidine component typically dosed at 100 mg a day.  In the US, while they can't be advertised for treatment of a disease, instead the FDA regards these constituent compounds as GRAS ("Generally Regarded As Safe") via FDA's published GRAS certifications on Orange Pomace and Orange Extract.

That was all regarding the oral ingestion route.  The inhalation route, of course, should be of particular interest for acute cases, but the downside risk is just this side of dicey that I'd prefer not to speculate on a public forum about it; suffice to say that if this OJ oral ingestion is found to have merit, I'd be happy to share with medical professionals the learnings I've assembled so far on the inhalation route. (Suffice to say that it doesn't involve orange juice).

So I close with a request: If you've gotten this far, I would really appreciate if you can poke holes in this analysis, and to particularly include same in the comments below so that others can evaluate.  In particular, I'd love a qualified answer to the question, "If these are qualified research paths, then which researchers are in the process of studying them?"

Please consider yourself warmly-welcomed to troll, call me a snake-oil peddler, or whatever, but just put some reasoning behind your concerns so that the reasoning can be shored up or thrown out as appropriate.  Disclaimer that I have no financial interest in any company, venture, or trading position associated with the above - I just want to get back to normal-life again.

- - - - -
tl;dr

Drink extra orange juice.  And don't gain a false sense of security from doing so.

Update 2 April: I 'professionalized' up this post into a Medium post that I'm feeling relieved has been getting attention from appropriate places: 
https://medium.com/@ricksher/with-the-coronavirus-epidemic-still-reaching-its-zenith-in-the-us-i-observe-friends-and-family-in-bff0e873fb5