A drug used to treat feline coronavirus infection may hold the key for developing a treatment for human COVID-19

0
443

University of Alberta researchers are racing against the clock to test an antiviral drug that has been proven to cure a cat coronavirus and is hoped to have the same effect on people with COVID-19.

“Our lab has been working as fast as we can to get our results out,” said biochemist Joanne Lemieux.

“We have not taken weekends, the days of the week have blurred. We’re all working non-stop to get results as fast as we can.”

The project is one of 11 at the U of A to receive funding from the federal government’s $52.6 million investment in COVID-19 research.

“There is a possibility that combination therapy can be used, so there are multiple lines of attack,” Lemieux said.

Following the worldwide outbreak of severe acute respiratory syndrome (SARS) in 2003, chemist John Vederas, biochemist Michael James (now a professor emeritus) and other U of A scientists studied a mechanism that stopped the virus from replicating in the laboratory.

The compounds, known as protease inhibitors, have since been further developed in the United States, tested and shown to also stop a fatal virus in cats.

Now Vederas, Lemieux and virologist Lorne Tyrrell are combining their labs’ efforts to test the inhibitor against the new coronavirus that is causing the worldwide COVID-19 pandemic.

“I’m very excited about this research project,” Lemieux said. “It’s nice to think that we can make a difference.”

How protease inhibitors work

It is estimated that five to 10 per cent of all new drugs in development worldwide are protease inhibitors.

They have been used successfully to target diseases including high blood pressure, congestive heart failure, HIV, Type 2 diabetes and even cancer.

COVID-19 is a ribonucleic acid (RNA) virus, as are many other infectious viruses such as Ebola, hepatitis C, West Nile and polio.

Proteases are enzymes that allow the virus to replicate inside a human host.

“When the virus enters a cell, the RNA is translated into a polypeptide – a long single protein chain – and the protease chops that long chain into many different parts, which then cause the damage,” explained Lemieux.

“If the protease does not work, the virus cannot replicate in the cell, so it’s a pretty clear antiviral target,” she said.

Vederas’ lab in the Faculty of Science will produce the inhibitor drug, and Lemieux’s lab will determine the crystal structure of the COVID-19 protease after it is blocked by the drug to observe how it works.

Tyrrell will test its effect against the viral load in a cell culture at his lab, which is federally approved to work with deadly pathogens such as COVID-19.

Connection to a cat virus

There are several promising things about this protease inhibitor that make the U of A researchers hopeful it will be a fit for COVID-19.

Genome sequencing of the novel coronavirus indicates that its protease is nearly identical (96 per cent) to the protease in the original SARS virus.

“Of the 306 amino acid residues in the chain that makes the 3CL protease of the ‘Wuhan’ virus, only 12 are different and they are highly similar in properties,” the researchers stated in their research proposal.

Another good sign is that a derivative of the same protease inhibitor was recently shown by American veterinary investigators to cure cats of feline infectious peritonitis, a coronavirus-caused condition that is almost always fatal to the animals.

“The key compound affected cures or significant remissions in all the cats,” the researchers stated.

”It is very exciting that the drug was effective and tolerated in cats,” said Lemieux, while cautioning that it still must be proven and tested in humans.

Translating discovery into life-saving products

Lemieux, who is director of the Membrane Protein Diseases Research Group within the U of A’s Faculty of Medicine & Dentistry, usually focuses her research on proteases associated with other diseases such as Parkinson’s and urinary tract infections, but all work in her lab has shut down except for the COVID-19 project.

“When I teach my classes at the university I try to impart that fundamental research can really assist us in drug development,” she said.

“I try to get the students excited about protein structures and protein chemistry, and especially how proteases can be inhibited for drug development.”

Tyrrell, who is the founding director of the Li Ka Shing Institute of Virology, said another advantage for the U of A project is that the institute has a commercialization hub designed to take promising bench research to patients as soon as possible through licensing or partnerships with pharmaceutical companies.

It is led by Michael Houghton, who identified the hepatitis C virus and has more than 70 patents in development.

Tyrrell said pharmaceutical companies can sometimes be reluctant to develop drugs against viruses that may be fleeting if they can be contained through public health measures, such as the SARS and MERS outbreaks. He said that may be different this time.

“With the crisis right now, it is critical that virologists translate some of the things we are discovering into products,” said Tyrrell.

Lemieux said the U of A researchers hope to know within the next two months whether the protease inhibitor they are developing is effective against the COVID-19 virus.

“Obviously cats and humans are different,” said Lemieux. “We’re far away from developing something to treat people, but I would call these promising first steps towards development of a protease inhibitor drug to treat either this outbreak or future ones.”


RESEARCH AND DEVELOPMENT IN SMALL MOLECULE ANTIVIRAL AGENTS FOR COVID-19 AND RELATED CORONAVIRUS DISEASES

Key Proteins and Their Roles in Viral Infection

Identification of targets is important for identifying drugs with high target specificity and/or uncovering existing drugs that could be repurposed to treat SARS-CoV-2 infection. 

Table 2 lists potential targets, their roles in viral infection, and representative existing drugs or drug candidates that reportedly act on the corresponding targets in similar viruses and thus are to be assessed for their effects on SARS-CoV-2 infection.

3CLpro and PLpro are two viral proteases responsible for the cleavage of viral peptides into functional units for virus replication and packaging within the host cells.

Thus, drugs that target these proteases in other viruses such as HIV drugs, lopinavir and ritonavir, have been explored.19 RdRp is the RNA polymerase responsible for viral RNA synthesis that may be blocked by existing antiviral drugs or drug candidates, such as remdesivir.19 

Conceivably, the interaction of viral S protein with its receptor ACE2 on host cells, and subsequent viral endocytosis into the cells, may also be a viable drug target. For example, the broad-spectrum antiviral drug Arbidol, which functions as a virus-host cell fusion inhibitor to prevent viral entry into host cells against influenza virus,20 has entered into a clinical trial for treatment of SARS-CoV-2.21,22 

The protease TMPRSS2 produced by the host cells plays an important role in proteolytic processing of S protein priming to the receptor ACE2 binding in human cells.11 It has been shown that camostat mesylate, a clinically approved TMPRSS2 inhibitor, was able to block SARS-CoV-2 entry to human cells, indicating its potential as a drug for COVID-19.11

Table 2

Key Proteins and Their Roles during the Viral Infection Process

target candidatefull namerole during viral infectiondrug candidate
3CLprocoronavirus main protease 3CLproa protease for the proteolysis of viral polyprotein into functional unitslopinavir19,30
PLpropapain-like protease PLproa protease for the proteolysis of viral polyprotein into functional unitslopinavir19,30
RdRpRNA-dependent RNA polymerasean RNA-dependent RNA polymerase for replicating viral genomeremdesivir,19,29,32 ribavirin16,29,31
S proteinviral spike glycoproteina viral surface protein for binding to host cell receptor ACE2Arbidol20,22,33a
TMPRSS2transmembrane protease, serine 2a host cell-produced protease that primes S protein to facilitate its binding to ACE2camostat mesylate11
ACE2angiotensin-converting enzyme 2a viral receptor protein on the host cells which binds to viral S proteinArbidol20,22,33a
AT2angiotensin AT2 receptoran important effector involved in the regulation of blood pressure and volume of the cardiovascular systemL-16349128

aAn inhibitor of viral entry to host cells. Its direct action on S protein and ACE2 is yet to be confirmed.

ACE2 involvement with coronavirus infection is of further interest since ACE2 is a potent negative regulator restraining overactivation of the renin-angiotensin system (RAS) that may be involved in elicitation of inflammatory lung disease in addition to its well-known role in regulation of blood pressure and balance of body fluid and electrolytes.23,24

 It catalyzes degradation of angiotensin II to angiotensin (1–7). The balance between angiotensin II and angiotensin (1–7) is critical since angiotensin II binds to angiotensin AT1 receptor to cause vasoconstriction, whereas angiotensin (1–7) elicits vasodilation mediated by AT2.2527 

Although the notion that ACE2 mediates coronavirus invasion is largely accepted, it remains unclear how the levels or activities of ACE2, AT1 receptors, and AT2 receptors are altered in coronavirus-induced diseases due to the limited number of studies.23,24 

Therefore, it is yet to be determined whether some drugs or compounds that target any of these proteins (e.g., L-163491 as a partial antagonist of AT1 receptor and partial agonist of AT2 receptor) may alleviate coronavirus-induced lung injury.28

Patents and Potential Drug Candidates Related to Key Protein Targets

The CAS content collection contains patents related to coronavirus key proteins listed above. Table 3 lists the number of patents related to each protein target and associated therapeutic compounds with a CAS Registry Number (CAS RN) reported in these patents. CAS data show that targets 3CLpro and RdRp attracted more attention than other targets, and more compounds with therapeutic potential were identified for these targets, probably due to the work done for SARS-CoV which also contains 3CLpro and RdRp.

Table 3

Key Protein Targets and Related Patents in the CAS Content Collection and Potential Drug Candidates in CAS REGISTRY of Chemical Substances

targetno. of patentsno. of potential drug candidates
3CLpro492178
PLpro4189
RdRp26570
S protein46333
ACE2597
AT2238

Existing Drugs with Potential Therapeutic Applications for COVID-19

Since SARS-CoV-2 is a newly discovered pathogen, no specific drugs have been identified or are currently available. An economic and efficient therapeutic strategy is to repurpose existing drugs.

On the basis of genomic sequence information coupled with protein structure modeling, the scientific community has been able to rapidly respond with a suggested list of existing drugs with therapeutic potential for COVID-19. 

Table 4 provides a summary of such drugs together with potential mechanisms of actions for their activities. Barcitinib was proposed because of its anti-inflammatory effect and possible ability to reduce viral entry.35 

A fixed dose of the anti-HIV combination, lopinavir–ritonavir, is currently in clinical trials with Arbidol or ribavirin.22 Remdesivir, developed by Gilead Sciences Inc., was previously tested in humans with Ebola virus disease and has shown promise in animal models for MERS and SARS. The drug is currently being studied in phase III clinical trials in both China and the USA.

Favipiravir, a purine nucleoside leading to inaccurate viral RNA synthesis,36 was originally developed by Toyama Chemical of Japan, and has recently been approved for a clinical trial as a drug to treat COVID-19.30 

Chloroquine, an antimalarial drug, has proven effective in treating coronavirus in China.32 

In addition to the above-mentioned, many other antiviral drugs are also listed.

Table 4

Existing Drugs with Therapeutic Potentials for COVID-19 (Drug Repurposing)

drug candidateCAS RNtargetpossible mechanism of action on COVID-19disease indication
baricitinib351187594-09-7JAK kinasea JAK inhibitor that may interfere with the inflammatory processesapproved drug for rheumatoid arthritis
lopinavir19a192725-17-0viral proteases: 3CLpro or PLproprotease inhibitors that may inhibit the viral proteases: 3CLpro or PLprolopinavir and ritonavir are approved drug combination for HIV infection
ritonavir19,37c155213-67-5   
darunavir33206361-99-1  approved drug for HIV infection
favipiravir (favilavir)29,36259793-96-9RdRpa purine nucleoside that acts as an alternate substrate leading to inaccurate viral RNA synthesisviral infections
remdesivir19,29,32a1809249-37-3 a nucleotide analogue that may block viral nucleotide synthesis to stop viral replicationEbola virus infection
ribavirin16,2931a36791-04-5  RSV infection, hepatitis C, some viral hemorrhagic fevers
galidesivir34b249503-25-1  hepatitis C, Ebola virus, Marburg virus
BCX-4430 (salt form of galidesivir)34b222631-44-9  hepatitis C, Ebola virus, Marburg virus
Arbidol22,33a131707-23-8S protein/ACE2dan inhibitor that may disrupt the binding of viral envelope protein to host cells and prevent viral entry to the target cellinfluenza antiviral drug
chloroquine29,3254-05-7endosome/ACE2a drug that can elevate endosomal pH and interfere with ACE2 glycosylationmalarial parasite infection
nitazoxanide2955981-09-4N/Aa drug that may inhibit viral protein expressionvarious helminthic, protozoal, and viral infection-caused diarrhea

aDrugs under clinical trials for treating COVID-19 (repurposing).bDrugs under clinical trials for other virus-induced diseases.cRitonavir is a pharmacokinetic profile enhancer that may potentiate the effects of other protease inhibitors due to its ability to attenuate the degradation of those drugs by the liver enzyme CYP3A4 and thus is used in combination with antivirial Lopinavir.37dAn inhibitor of viral entry to host cells. Its direct action on S protein and ACE2 is yet to be confirmed.

Selected Patents Related to Promising Small Molecule Drug Candidates

Table 5 shows selected patents associated with the aforementioned potential drugs, together with patents disclosing small molecules for treatment of SARS or MERS.

The selection was based on the presence of important terms in CAS-indexed patents as well as the presence of the synthetic preparation role assigned by CAS scientists during document indexing.

Patent applications WO2009114512 and WO2014028756 disclose preparation of compounds active as JAK inhibitors, one of which was later named as baricitinib and developed for reducing inflammation in rheumatoid arthritis.

Patent application JP5971830 discloses preparation of polycyclic pyridone compounds and their use as endonuclease inhibitors. Patent applications US20160122374 and US20170071964 disclose preparation of the nucleotide analog drug remdesivir that was later developed as a therapeutic agent for Ebola and Marburg virus infections (Patent US20170071964).

Because of its promising results in at least two COVID-19 patients, remdesivir has now entered into phase III clinical trials.

Table 5

Selected Patents Associated with Potential Drugs (Repurposing) for COVID-19 or Small Molecules for Treatment of SARS or MERS

patent no.priority datetitleorganization
WO200911451220080311Preparation of azetidine and cyclobutane derivatives as JAK inhibitorsIncyte Corporation, USA
WO201402875620140220Deuterated baricitinibConcert Pharmaceuticals, Inc., USA
JP597183020150428Preparation of polycyclic pyridone derivatives as cap-dependent endonuclease (CEN) inhibitors and prodrugs thereofShionogi and Co., Ltd., Japan
US2016012237420141029Preparation of nucleosides and methods for treating Filoviridae virus infectionsGilead Sciences, Inc., USA
US2017007196420160916Preparation of amino acid-containing nucleotides and methods for treating arenaviridae and coronaviridae virus infectionsGilead Sciences, Inc., USA
WO200707514520070704Preparation of benzopyranone derivatives as anti-coronaviral agentsSingapore Polytechnic, Singapore; Shanghai Institute of Materia Medica Chinese Academy of Sciences, China
WO200502151820050310Preparation of 3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid derivatives as cysLT2 receptor antagonists for treatment of respiratory diseasesOno Pharmaceutical Co., Ltd., Japan
WO200712016020071025Preparation of N-heterocyclic acetamides useful for viral inhibitionNovartis AG, USA
WO200911916720091001Aniline derivative having anti-RNA viral activityKinoPharma, Inc., Japan
WO201304938220130404Broad-spectrum antivirals against 3c or 3c-like proteases of picornavirus-like supercluster: picornaviruses, caliciviruses and coronavirusesKansas State University Research Foundation; The Ohio State University; Wichita State University – all in USA
WO201804234320180308Preparation of peptides that inhibit 3C and 3CL proteases and methods of use thereofGlaxoSmithKline, UK
WO200706751520070614Five-membered iminocyclitol derivatives as selective and potent glycosidase inhibitors: new structures for antivirals and osteoarthritis therapeuticsAcademia Sinica, Taiwan

Patent application WO2013049382 discloses both structures and syntheses of compounds from various structure classes (peptidyl aldehydes, peptidyl α-ketoamides, peptidyl bisulfite salts, and peptidyl heterocycles), as well as certain formulation compositions, developed to inhibit viral 3C protease or 3C-like protease (i.e., 3CLpro).

Patent application WO2018042343 presents both preparation methods and biological assay results for compounds capable of inhibiting the SARS virus proteases. These compounds appeared to exhibit good enzyme-inhibiting activity (pIC50 ≈ 7 or IC50 ≈ 0.1 μM) and antiviral activity, which was assessed by host cell viability using cultured human lung fibroblast MRC-5 cells infected with a specified virus (e.g., MERS virus) expressing the viral S protein. Drug administration routes were also mentioned in this patent.

Small Molecule Compounds in Research and Development with Potential Effects on Key Protein Targets for Human Coronavirus-Induced Diseases

Besides various commercialized antiviral drugs, there are also small molecule compounds currently in research and development that have shown significant inhibitory effects on many key proteins from similar coronaviruses such as SARS-CoV and MERS-CoV (Table 6).

These drug candidates mostly inhibit viral enzymes including proteases and components for RdRp. Since 3CLpro protease has a high level of sequence homology between SARS-CoV and SARS-CoV-2, inhibitors against 3CLpro of SARS-CoV may also be applicable to SARS-CoV-2.

Compounds, including benzopurpurin B, C-467929, C-473872, NSC-306711 and N-65828, which may inhibit the activity of viral NSP15, poly(U)-specific endoribonuclease, were tested for reduced SARS-CoV infectivity in cultured cells with IC50 of 0.2–40 μM.38 

Compound C-21 and CGP-42112A are two AT2 agonists, whereas L-163491 has dual functions as a partial agonist for AT2 receptor and a partial antagonist of AT1 receptor. Since AT1 and AT2 are important effectors in the RAS system to which ACE2 belongs, it has been speculated that these compounds may be used to adjust the balance between AT1 and AT2, which may be affected by coronavirus infection and to alleviate viral-induced lung injury during the infection.24

Table 6

Small Molecule Compounds in Research and Development with Therapeutic Potential for COVID-19

CAS RNsmall molecule compoundtargetpossible mechanism of action on COVID-19
4431-00-9aurine tricarboxylic acidRNA-dependent RNA polymerase (RdRp)an inhibitor that may bind to viral RdRp, as tested against SARS-CoV in cell culture16
502960-90-94-methyl-N-[(1S)-2-oxo-2 [[(1S,2E)-1-(2-phenylethyl)-3-(phenylsulfonyl)-2-propen-1-yl]amino]-1-(phenylmethyl)ethyl]- 1-piperazinecarboxamideviral proteases: 3CLpro and PLproan inhibitor that may disrupt the function of 3CLpro and PLpro, which was tested against SARS-CoV16,39,40
1851279-09-84-(1,1-dimethylethyl)-N-[(1S)-2-oxo-2-[[(1S,2E)-1-(2-phenylethyl)-3-(phenylsulfonyl)-2-propen-1-yl]amino]-1-(phenylmethyl)ethyl]- 1-piperazinecarboxamide  
1851280-00-64-(2-methoxyethyl)-N-[(1S)-2-oxo-2-[[(1S,2E)-1-(2-phenylethyl)-3-(phenylsulfonyl)-2-propen-1-yl]amino]-1-(phenylmethyl)ethyl]- 1-piperazinecarboxamide  
223537-30-2rupintrivir a cysteine protease inhibitor that may disrupt the function of 3CLpro and PLpro41
2409054-43-7R)-α-[[3-(4-chloro-2-fluorophenyl)-1-oxo-2-propen-1-yl]amino]-N-[(1R)-1-methyl-2-(2-oxo-3-pyrrolidinyl)ethyl]- benzenepropanamideviral proteases: 3CLpro or PLproan inhibitor that may disrupt the function of 3CLpro or PLpro, as tested against SARS-CoV or MERS-CoV39,40
452088-38-95-[(4-methyl-1-piperidinyl)sulfonyl]-1H-indole-2,3-dione  
2409054-44-83-hydroperoxy-4-[2-hydroxy-3-[3-(4-hydroxyphenyl)-1-oxo-2-propen-1-yl]-6-methoxyphenyl]-2-butanone  
41137-87-5hirsutenone  
992-59-6benzopurpurin BNSP15 (poly(U)-specific endoribonuclease)chemical inhibitors that may suppress viral infectivity by inhibiting endoribonuclease NSP15, as tested against SARS-CoV in cultured cells38
351891-58-2C-467929  
331675-78-6C-473872  
813419-93-1NSC-306711  
501444-06-0N-65828  
477775-14-7C-21AT2an angiotensin AT2 receptor agonist that may alleviate the virus-induced lung injury24
127060-75-7CGP-42112A  
170969-73-0L-163491 a dual-property molecule that functions as angiotensin AT1 partial antagonist and AT2 agonist which may alleviate the virus-induced lung injury24

Small Molecules Identified by Structure Similarity, Lipinski’s Rule of 5, and CAS-Indexed Pharmacological Activity and/or Therapeutic Usage

Besides the aforementioned antiviral drugs, there may be additional small molecule compounds with therapeutic or pharmacological potential against viruses such as SARS-CoV and MERS-CoV. Compounds listed in Tables 4 and 6 were subjected to a Tanimoto similarity search in CAS REGISTRY using CAS proprietary fingerprints.a 

Those substances with at least 60% structural similarity match and meeting Lipinski’s rule of 5 were identified. Table 7 lists selected compounds that were also identified to have a pharmacological activity or therapeutic usage role. Compound name and CAS RN are provided for each compound.

The second column lists the number of compounds that met the structure similarity and Lipinski’s rule criteria. Although more work remains to be done in this regard, the methodology and results mentioned here point to a strategy that may help streamline the process of drug discovery for COVID-19.

Table 7

Examples of Similar Molecules with Possible Therapeutic Effects Identified by Structural Similarity, Lipinski’s Rule of 5, and Pharmacology/Therapeutic Role Assigned by CAS Scientists during Document Indexing

query substance name (CAS RN)no. of substances with >60% similarityexample of selected similar substanceRegistry Number of selected similar substance
ribavirin (36791-04-5)1520viramidine119567-79-2
galidesivir (249503-25-1)502(2R,3S,5R)-5-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-3-hydroxy-2-pyrrolidinemethanol1610426-50-0
  (2S,4R,5S)-5-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-4-hydroxy-2-pyrrolidinemethanol872534-76-4
  (2R,3R,4S,5S)-5-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-3-hydroxy-4-methoxy-2-pyrrolidinemethanol1610426-51-1
chloroquine (54-05-7)21176hydroxychloroquine118-42-3
  (±)-chloroquine diphosphate50-63-5
  chloroquine hydrochloride3545-67-3
  chloroquine sulfate132-73-0
favipiravir (259793-96-9)3096-bromo-3,4-dihydro-3-oxo-2-pyrazine-5-d-carboxamide1476773-04-2

Source:
University of Alberta

LEAVE A REPLY

Please enter your comment!
Please enter your name here

Questo sito usa Akismet per ridurre lo spam. Scopri come i tuoi dati vengono elaborati.