Protein Cell: The Li Honglin/Xu Ke team found powerful anti-new crown virus candidates and clinically available drugs targeting host DHODH.

As a new concept anti-new crown virus drug, the inhibitor of dihydro whey acid dehydrogenase (DHODH) dehydrogenase (DHODH) was fully funded by LifeArc UK, a leading public interest research institution in the field of pharmaceutical innovation, on 29 May 2020. Professor Li Honglin, who co-directed the International Multi-Center Clinical Trials (DEFEAT-COVID study) with UK clinical experts, is the initiator and lead scientist of the clinical trial and has been approved for clinical trials by the UK Medicines and Healthcare Products Authority (MHRA) to begin recruiting COVID-19 patients at Ashford and St Peters Hospital.
" SARS-CoV-2, SARS-CoV, Zika, MERS-CoV, Ebola, H7N9 and other RNA viruses in local areas / worldwide large-scale outbreak epidemic, bringing serious harm to people's lives and health, but also to the country and the world's economic development has a huge impact.
so far, the new corona virus has infected 20 million people worldwide and killed 730,000 people, but no special vaccines or antiviral drugs have been developed.
virus, the search for effective, low-toxic drugs to treat the new coronary pneumonia is still the world's top priority in the fight against the new coronary virus.
August 4, 2020, The Li Honglin Team of the School of Pharmacy of East China University of Technology/Shanghai New Drug Design Key Experiment and Xu Ke team of the National Key Laboratory of Virology of Wuhan University published a study on "Novel and Potent bondedors targeting DHODH are broad-spectrum antivirals against RNA viruses including new-found-syned coronaviru" s SARS-CoV-2" reported that dehydrogenase dehydrogenase (DHODH) inhibitors targeted at host cells can perform broad-spectrum antiviral effects by inhibiting viral genome replication and regulating immunity, and that two new DHODH inhibitors S312 and S416 have shown efficient anti-new crown virus activity at the cellular level. Among them, S416 is the most active candidate for anti-new coronary virus in in-body inhibition, which has been reported so far.
replication cycle of the virus consists of five steps: adsorption, perforation, shelling, nucleic acid and protein synthesis, assembly, and release.
can be divided into targeted viruses (Direct Acting Antivirals Agent, DAA) and targeted host cells (Host-Targeting Antiviral, HTA) depending on the object of the existing antiviral drug action.
DAA drugs targeted at viral particles are drugs designed for one or more processes of the virus replication cycle, such as the drug Abidor, which targets influenza virus membrane adsorption fusion, the drug Enfverdi and chloroquine, the drug targeting coronavirus (CoV) S protein, the drug GLS-5300, the drug that acts as a virus shell release nucleic acid stage of the aluminoid anti-flu virus drugs, Drugs that act on viral nucleic acid replication stages (e.g. retrovirase inhibitor Ramifedin, integrated enzyme inhibitor Latravir, RNA-dependent RNA polymerase (RdRp) inhibitors Fapilave and Redsivir), drugs that act on viral protein translation and assembly stages such as HIV protease inhibitor Lopinavir, drugs that act on the release stage of mature sub-viruses, such as influenza virus neuroticase inhibitor osethane inhibitor otamivir, etc.
Because of the virus-specific nature of DAA drugs, existing DAA drugs have limited or completely ineffective treatment of new viruses (such as Abidol, chloroquine, fapilave, lopinavir and Redsivir treatment COVID-19 is not satisfactory clinical performance), and the re-development of new DAA drugs is also "far from quenching thirst."
, HTA drugs that target host factors have obvious advantages in controlling new virus outbreaks.
Virus is a parasitic organism, must rely on the host cell raw materials and machines for nucleic acid and protein production and processing to complete replication, HTA drugs can not only effectively inhibit the rapid replication of viral nucleic acids, but also to fight viral resistance mutations, so as to "unchangeable."
therefore, the development of HTA drugs with broad-spectrum antiviral effects has been the goal pursued in the field of research and development of new anti-infection drugs.
however, because such drugs are targeted at the host, more consideration needs to be given to safety and in vitro-in vivo drug conversion problems, therefore, the development of such drugs often do more than half the effort, less effort.
base is an essential raw material for viral RNA/DNA replication, HTA drugs that target the synthesis pathway of nicosine nucleotides are the focus of research on broad-spectrum antiviral drugs in recent years.
For the synthesis pathway of nihonucleotides, although some active compounds are reported, but most of them do not have a clear target, even if the target is clear, but also basically in the animal body invalid or the effect is not significant and fold sand.
Dihydro whey acid dehydrogenase (DHODH), located on the outer side of the membrane in the mitochondria, is a key enzyme in the fourth step of the oxidation reaction of catalytic nicotin nucleotides from the head synthesis pathway, and those rapidly proliferating cells (lymphocytes, tumor cells, etc.) or virus replication require a large amount of synthesis from the head synthesis pathway Nirone nucleotides meet the need for cell proliferation or viral replication of nucleic acids, so that the activity of DHODH can be inhibited by inhibiting the starting synthesis of the nihoma base, thereby inhibiting the rapid proliferation of lymphocytes/tumor cells or virus DNA and RNA biosynthesis, playing an anti-autoimmune disease, cancer and antiviral role.
The inhibitors targeted at DHODH currently have only two FDA-approved drugs on the market, fluoromete and Trifroamine (i.e., A771726, the active metabolite in the body of drefomit), for the treatment of rheumatoid arthritis and multiple sclerosis, respectively.
For DHODH targets, no new drugs have been on the market for more than 20 years, and although new DHODH inhibitors have been reported in recent years, most of them lack inhibition of DHODH or fail to show significant therapeutic effects on animal models, limiting their further development and use.
DHODH target has the potential to develop broad-spectrum anti-RNA viruses, the feasibility of the application of inhibitors in broad-spectrum antivirals in the body has been unresolfected.
Li Honglin team and Xu Ke team after many years of in-depth cooperation, joint research, in the target host of broad-spectrum antiviral candidate drug research has made important progress.
Li Honglin's team developed a variety of drug design methods in the early stage, and designed two new powerful candidate drugs S312 and S416 for DHODH targets, which inhibited the IC50 value of DHODH at the molecular level. At 29.2 nM and 7.5 nM, respectively, the KD values combined with DHODH were 20.3 nM and 1.69 nM, respectively, and showed good drug-taking properties such as fast binding and slow dissocionation.
team, this paper validates that both drugs and the existing DHODH inhibitors, Leflunomide and Teriflunomide, have shown broad-spectrum antiviral activity against a variety of RNA viruses, including influenza A (H1N1). H3N2, H9N2), Zika and Ebola viruses (cell level EC50 range 0.013 to 35.02 ?M), and S312 and S416 in-body antiviral effects are superior to Triflamide.
in the process of writing this article, just in time for the outbreak of the new crown epidemic in China, based on the previous research results and mechanism of action analysis, the team quickly responded to carry out the active test against the new crown virus.
results showed that the two candidate drugs, S416 and S312, were 0.017 ?M (SI and 10505.88) and 1.56 ?M (SI?gt;101.41), respectively.
S416 cell test antiviral effect than Redsiwe (EC50 s 0.77 sm, SI sgt; 129.87) is 45 times stronger and more than 60 times more active than chloroquine (EC50 s 1.13 sm, SI sgt; 88.5), which is the most active anti-SARS-CoV-2 candidate compound reported to be inosome.
existing older drug fluorometer and active metabolite Trifroamine, EC50 effective against SARS-CoV-2 virus in in-body (EC50 s 6.001 sm, moi s 0.03; 26.06 sm, moi s 0.05), excellent More than 2 times more than fapilave (EC50 s 66.85 sm, moi s 0.05) and in clinical cases, if the first day of the drug is given at a low dose of 10 mg/day defluoromete can maintain a high blood drug treatment concentration, suggesting that the old drug fluoromet or Trifroamine may have better clinical application potential against the new corona virus.
In order to verify the feasibility of the application of DHODH inhibitors in broad-spectrum antiviral applications in the body, the paper adopts experimental design strategies ranging from in vivo drug effectiveness verification to DHODH gene knockout to quantitative detection of viral genome replication, to the resupply of different raw materials required for genome replication, and finally confirms DHODH as an effective therapeutic target for broad-spectrum anti-RNA virus in animals for the first time.
used animal models of influenza A virus infection, animal models infected with the drug-resistant strain NAH275Y of Oseltamivir, and animal models of infection with the insensitive strain SC09 of Oseltamivir to confirm that S312 not only has the same efficacy as DAA drugs, but also has an advantage in combating DAA drug-resistant viruses.
Using CRISPR/Cas9 gene knockout technology to establish the DHODH-/-A549 cell line, it was found that the growth of the H1N1 influenza virus on the DHODH-/-cell strain was significantly lower than that of the WT cell strain and did not affect the normal growth of cells, proving that DHODH is an important host enzyme required for influenza virus replication.
using the influenza virus micro-replication subsyste system to quantify viral genome replication, S312 and S416 were found to play a broad-spectrum antiviral role by inhibiting viral RNA replication.
add A, G, U, C four nucleotides to observe its effect on influenza virus rescue, found that only adding U and C instead of A and G can save influenza virus replication inhibited effect, indicating that S312 targeted synth synthase; Dehydro whey acid (DHO) and product whey acid (ORO), found that ORO instead of DHO added dose dependence to save inhibited influenza virus replication, indicating that S312 by targeting DHODH, inhibit the replication of the virus genome.
DHODH is a key enzyme in the synthesis pathway of pyridine from the beginning, so the antiviral activity of the drug will be further enhanced if the DHODH candidate drug found in this project is used in association with the inhibitor of the pyridine remediation pathway.
Went on, the paper confirms the therapeutic effect of S312 over Oseltamivir in severe mice infected with influenza virus, and then suggests that the combination of DAA and HTA is an effective strategy for antiviral therapy.
in the middle and late stages of viral infection (5-9 days after infection), Duffy has lost treatment, while the S312 can still play a 50% role in mouse rescue, if combined with Duffy can play a 100% rescue effect.
, such as influenza, new coronary pneumonia in the late stages of infection in the body will produce an excessive inflammatory response and thus induce a "cytokine storm."
article tested the albatross washes of diseased mice before and after the use of Duffy and S312 on the 14th day after influenza virus infection in mice, and found that DHODH inhibitors reduced IL6, MCP-1, IL5, KC/GRO (CXCL1), IL2, The levels of inflammation factors such as IFN-10, IP-10, IL9, TNF-alpha, GM-CSF, EPO, IL12p70, MIP3 alpha and IL17A/F not only effectively cut off nucleic acid sources to inhibit viral replication, but also inhibit the expression and release of excessive inflammatory factors, playing an antiviral and anti-inflammatory effect in the late stages of infection.
This paper not only found and verified that the target host cell DHODH inhibitors can play a broad-spectrum antiviral effect by inhibiting viral replication and regulating immunity, but also suggests that the old drug lefromet or Trifluoride may have better clinical application potential of anti-new coronavigic virus, which can meet the current clinical needs of new coronavigic pneumonia.
, the newly designed candidate drugs S312 and S416 are also expected to be developed as candidates for anti-RNA viruses, providing a reserve for future battles against acute infectious diseases of coronaviruses and other RNA viruses.
At present, the old drug fluoromet has been conducted in Wuhan University People's Hospital on the new coronary pneumonia (COVID-19), a multi-center, randomized, double-blind, controlled clinical trial for moderate, severe patients (clinical registration number: ChiCTR2000030058). The results of sympathetic drug use in the early part of
showed that the detox time (median 5 days) of patients with oral fluoromet was significantly shorter than that of control patients under the same treatment conditions (median 11 days), with a statistically significant difference of p.0046.
, the level of acute inflammatory C-reactive protein decreased significantly in patients in the drug group, which confirmed the antiviral and anti-inflammatory effects of the drug.
addition, as a new concept anti-new coronary virus drug, fluoromet received full funding (1.5 million) from LifeArc UK, a leading public interest research organization in the field of pharmaceutical innovation, on 29 May 2020. Professor Li Honglin, who co-directed the International Multi-Centre Clinical Trials (DEFEAT-COVID study) with BRITISH clinical experts, is the initiator and lead scientist of the clinical trial and has been approved for clinical trials by the UK Medicines and Healthcare Products Authority (MHRA).