ENZYMATIC TARGETS
The enzymatic target of GC376 is the main protease (Mpro) of SARS-CoV-2 [1]. The Mpro of SARS-CoV viruses has been well documented and studied since the early outbreak of SARS-CoV in the early 2000s [2]. We know that viral proteases are prime targets of choice because of the ability for the drug of interest to prevent additional formation of viral proteins essential for continuation of the proliferation of viral cells [2]. We know that the Mpro of SARS-CoV-2 cleaves sequences after reading a glutamine residue, and because of this specificity, makes Mpro a great enzyme of interest for future drug targets [2]. The reason for the GC376’s target is because the Mpro is essential towards viral replication through cleavage of the virus’ polypeptide chain [1]. This is essential because it ultimately prevents correct proteins from being made which then impacts overall protein function and structure of viral envelope through modification of Cys145. Through cleavage of the polypeptide chain, the virus can no longer replicate proteins essential in the viral replication pathway as shown below.
The inhibition of Mpro is essential to stop the formation of structural proteins essential for viral replication and stability through interrupting the process peptide bond cleavage. Creating a target for inhibition of the catalytic cysteine residue is essential in preventing SARS-CoV-2 from replicating. While there is minimal evidence to support short-term drug resistance, there was a concern that long-term resistance could be acquired [4]. In almost 20 in vitro passages there was no indication of resistance to treat a feline coronavirus [4]. Future work to conduct studies on potential resistance and or ability for peptide mutations to counteract GC376 must be completed to help gain a stronger understanding of potential properties of SARS-CoV-2 in response to GC376.
ENZYME MECHANISM
The main proteases of the SARS coronaviruses (Mpro) cleave two overlapping polyproteins encoded by the viral RNA: pp1a and pp1ab. They cleave pp1a at 11 sites that contain a canonical Leu-Gln-(Ala/Ser) sequence between the Gln and Ala/Ser residues. The active site of Mpro is made of Cys145 ion paired with His41 to form a catalytic dyad, with an additional water molecule occupying the position of Asp from a typical Ser-His-Asp triad [5]. Surrounding the active site is an oxyanion hole consisting of Gly143, Ser144, and Cys145 [6]. The canonical sequence of the peptide substrate binds at the enzyme active site to form a Michaelis Complex. Acylation of Cys145 releases the N-terminal polypeptide and creates a C-terminal substrate/thiol group moiety. Deacylation of Cys145 regenerates the thiol and releases the C-terminal polypeptide. This step is rate limiting Water may stabilize the imidazolium ring intermediate [5].
INHIBITOR MECHANISM OF ACTION
GC376 is a prodrug and quickly converts to GC373 when incubated with SARS-CoV-2 Mpro. The benzyl ring and γ-lactam of the glutamine surrogate in the P1 position of GC373, as seen on the right, form a stacked hydrophobic interaction that serves to stabilize the inhibitor in the active site. P2 inserts into a hydrophobic pocket, and the carbonyl of P3 forms a hydrogen bond with the backbone amide of Glu166 [6].
The antiviral is a viral protease transition state inhibitor designed for protease 3C/3CL [1], an important protease in FIPV, a feline coronavirus [7]. This protease and subtle variants of it have been found in other coronaviruses as well, including SARS-CoV-2[8], making it a broad-spectrum coronavirus antiviral [9]. GC376 is a reversible, competitive inhibitor. Cys145 as such binds reversibly to the “C-terminus” of the protein mimic, with the mesylate group converting to an aldehyde within the cell and the resulting electrophilic terminal carbon being an ideal target for the Cys145 sulfhydryl. The final product is a hemithioacetal on the terminal carbon [1]. The leucine residue is important for hydrophobic pocket interactions with residues on the protease chain. There is extensive hydrogen bonding between the benzene-side ester, the pyrrolidone, and the business end carbonyl [9]. On the protease, C145 binds covalently to the inhibitor. G143, S144 and C145 support the oxyanion hole, while H163 and E166 support the chain through hydrogen bonding [1].
Some residues are a bit different in this study, it’s an analysis of PEDV not FIP. Much of the capability of the drug, however, is conserved [9]. The coronavirus spike proteins have much in common and this drug seems to be able to act on many of them.
Many different amino acids were considered for the second spot, as 3CLpro is a promiscuous protein that is capable of acting on many different target sequences [12]. After testing, Leucine performed the best in the second spot with Methionine and Isoleucine making 2nd and 3rd [9].
DRUG DEVELOPMENT
Before developing GC376, the authors first synthesized a series of protease inhibitors originally meant for treatment of norovirus infection. Because the norovirus 3C protease is essential to viral replications, William C. Groutas and colleagues [13, 14] designed several orally administrable functionalized dipeptide and tripeptide aldehyde transition state inhibitors of the 3C protease.
Each variation has the same recognition element but a different warhead. GC373 is a dipeptidyl aldehyde, GC375 is an α-ketoamide, and GC376 is a bisulfite adduct. GC376 was developed by reacting GC373 with sodium bisulfite (NaHSO3) [15]. However, several studies [1, 15] have suggested that GC376 actually acts as a prodrug of GC373, because x-ray crystal structures show that the bisulfite of GC376 is converted back to an aldehyde. This aldehyde functional group is then able to form reversible covalent bonds with the proteases to inhibit their function [15].
In vitro assays showed that both GC373 and GC376 strongly inhibited viral replication or viral protease activity for most viruses tested, except for HAV and FCV [15]. The space constraints in the protease of HAV or FCV could explain the minimal activity of these three variations against HAV and FCV; sequence analysis of FCV proteases showed a preference for small amino acids such as Ser or Ala in the critical S2 site rather than bulkier amino acids. GC375 was as potent or more potent than GC373 and GC376 against most of the viruses tested [15]. However, its relative inactivity against certain viral proteases is thought to be because its ketoamide group is too bulky to fit into protease active sites.
The use of the GC376 prodrug rather than similar alternatives could be advantageous in overcoming physiological obstacles associated with the aldehyde functional group of GC373 and steric hindrances associated with the ketoamide group of GC375.
ANIVIVE LIFESCIENCES STUDIES
According to the Anivive Site GC376 is in the mid stages of the development project- focusing on scaling manufacturing and further testing from the original intended use of the molecule in fighting Feline Infectious Peritonitis Virus (FIPV). Researchers have noticed the possibility of using GC376 as a potential treatment as the company states, they found it to be stable in human plasma, highly active against Sars-CoV-2 in relevant doses, and are currently preparing for phase 1 clinical testing. Most current research focuses on Feline Infectious Peritonitis Virus (FIPV) and how GC376 inhibits 3CLpro function.
In the university study, Kim et al. 2016 there was an improvement in health 2 weeks after starting treatment once infected with FIP (Feline Infectious Peritonitis), a type of coronavirus that uses the same 3C-like protease (3CLpro) to break up its polyprotein needed for reproduction [16]. In this study, cats were experientially infected with Feline Infectious Peritonitis caused by the Feline Infectious Peritonitis Virus (FIPV), a mutated Feline enteric coronavirus (FECV) is 100% fatal once certain symptoms appear. Once antiviral treatment with GC376 was started, there was a rapid improvement in condition and eventual full recovery. They found GC376 to inhibit the function of the 3CLpro protease, which is conserved among other coronaviruses including SARS. Treatment with the antiviral resulted in “significant reduction” in viral load in the cats, and that it was well tolerated and safe [16].
In Pedersen et al. 2018, researchers performed a field experiment using 20 cats, with various symptoms of FIP, they tested the effects of the GC376 of treating the disease. They found the 19 out of the 20 cats regained outward health within 2 weeks of treatment [4]. Relapse did occur in 13 of the 19 cats that were unable to be treated with the inhibitor, which may be caused by the different forms and manifestations of FIP allowed in the study as well as the differing ages of the cats. They concluded that GC376 was a promising treatment for certain presentations of FIP, but not all. The best inhibition of 3CLpro in reducing viral load was with naturally occurring FIP outside the central nervous system, while sustained remissions depended on various factors [4].
Both studies showed promises of GC376 as an inhibitor for coronaviruses, demonstrated with FIP experiments. When tested on cats experimentally infected with FIP the results were better than when a field study including 20 cats with various forms of FIP was performed, which was the more recent of the two studies. Both show promise of inhibiting 3CLpro used by coronavirus but more testing is needed; however, both studies did find the inhibitor to be safe and well tolerated, making it a great candidate for potential treatment of SARS-CoV-2.
CURRENT STATE OF DRUG USE
Recent studies and drug trials with feline subjects have used GC376 as a single agent to analyze activity in vitro and in vivo. Though it was produced to be orally administrable, in animal studies, GC376 has been administered subcutaneously in a highly pure form suspended in 10% ethanol and 90% polyethylene glycol 400 [4]. Upon further development, Anvive plans to use GC376 as a potential addition to the current standard treatments for COVID-19 [17]. Combination therapies have been widely used to combat viral infection because targeting several essential viral enzymes is typically more effective than targeting a single enzyme. Specifically with SARS-CoV-2, researchers have suggested combining antiviral treatments such as GC376 with other antiviral treatments and anti-inflammatory treatments [18]. This way, a single treatment regimen could directly inhibit viral enzymes while also dampening a potentially overactive host inflammatory response.
REFERENCES
Vuong, W., Khan, M.B., Fischer, C. et al. (2020) Feline coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication. Nature Communications 11, 4282. https://doi.org/10.1038/s41467-020-18096-2
Ullrich, S., & Nitsche, C. (2020). The SARS-CoV-2 main protease as drug target. Bioorganic & Medicinal Chemistry Letters, 30(17), 127377. https://doi.org/10.1016/j.bmcl.2020.127377
SARS-CoV-2 and COVID-19 Pathogenesis: A Review. (2020). Retrieved November 06, 2020, from https://www.lsbio.com/media/whitepapers/sars-cov-2-and-covid-19-pathogenesis-a-review
Pedersen, N. C., Kim, Y., Liu, H., Galasiti Kankanamalage, A. C., Eckstrand, C., Groutas, W. C., ... & Chang, K. O. (2018). Efficacy of a 3C-like protease inhibitor in treating various forms of acquired feline infectious peritonitis. Journal of Feline Medicine and Surgery, 20(4), 378-392. https://doi.org/10.1177/1098612X17729626
Ma, C., Sacco, M.D., Hurst, B. et al. (2020) Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease. Cell Research 30, 678–692. https://doi.org/10.1038/s41422-020-0356-z
Hung, H. C., Ke, Y. Y., Huang, S. Y., Huang, P. N., Kung, Y. A., Chang, T. Y., ... & Tsai, Y. R. (2020). Discovery of M Protease inhibitors encoded by SARS-CoV-2. Antimicrobial Agents and Chemotherapy, 64(9). https://doi.org/10.1128/AAC.00872-20
Update on GC376 and EVO984/GS441524 and when they might be available to treat FIP. (2020, August 20). Retrieved October 29, 2020, from https://www.zenbycat.org/blog/update-on-gc376-and-evo984-gs441524-and-when-they-might-be-avaialbe-to-treat-fip
Muramatsu, T., Takemoto, C., Kim, Y. T., Wang, H., Nishii, W., Terada, T., ... & Yokoyama, S. (2016). SARS-CoV 3CL protease cleaves its C-terminal autoprocessing site by novel subsite cooperativity. Proceedings of the National Academy of Sciences, 113(46), 12997-13002.
Ye, G., Wang, X., Tong, X., Shi, Y., Fu, Z. F., & Peng, G. (2020). Structural Basis for Inhibiting Porcine Epidemic Diarrhea Virus Replication with the 3C-Like Protease Inhibitor GC376. Viruses, 12(2), 240. https://doi.org/10.3390/v12020240
Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., ... & Bourne, P. E. (2000). The protein data bank. Nucleic acids research, 28(1), 235-242. https://doi.org/10.1093/nar/28.1.235
St. John, S., Anson, B. & Mesecar, A. (2016) X-Ray Structure and Inhibition of 3C-like Protease from Porcine Epidemic Diarrhea Virus. Scientific Reports 6, 25961. https://doi.org/10.1038/srep25961
Kim, Y., Mandadapu, S. R., Groutas, W. C., & Chang, K. O. (2013). Potent inhibition of feline coronaviruses with peptidyl compounds targeting coronavirus 3C-like protease. Antiviral Research, 97(2), 161–168. https://doi.org/10.1016/j.antiviral.2012.11.005
Tiew, K. C., He, G., Aravapalli, S., Mandadapu, S. R., Gunnam, M. R., Alliston, K. R., Lushington, G. H., Kim, Y., Chang, K. O., & Groutas, W. C. (2011). Design, synthesis, and evaluation of inhibitors of Norwalk virus 3C protease. Bioorganic & Medicinal Chemistry Letters, 21(18), 5315–5319. https://doi.org/10.1016/j.bmcl.2011.07.016
Mandadapu, S. R., Weerawarna, P. M., Gunnam, M. R., Alliston, K. R., Lushington, G. H., Kim, Y., Chang, K. O., & Groutas, W. C. (2012). Potent inhibition of norovirus 3CL protease by peptidyl α-ketoamides and α-ketoheterocycles. Bioorganic & Medicinal Chemistry Letters, 22(14), 4820–4826. https://doi.org/10.1016/j.bmcl.2012.05.055
Kim, Y., Lovell, S., Tiew, K. C., Mandadapu, S. R., Alliston, K. R., Battaile, K. P., ... & Chang, K. O. (2012). Broad-spectrum antivirals against 3C or 3C-like proteases of picornaviruses, noroviruses, and coronaviruses. Journal of Virology, 86(21), 11754-11762. https://doi.org/10.1128/JVI.01348-12
Kim Y, Liu H, Kankanamalage ACG, Weerasekara S, Hua DH, et al. (2016) Correction: Reversal of the Progression of Fatal Coronavirus Infection in Cats by a Broad-Spectrum Coronavirus Protease Inhibitor. PLOS Pathogens 12(5): e1005650. https://doi.org/10.1371/journal.ppat.1005650
Anivive Repurposes Veterinary Drug GC376 for COVID-19 And Submits Pre-IND to FDA. (2020, May 26). Retrieved October 29, 2020, from https://www.anivive.com/news/anivive-repurposes-veterinary-drug-gc376-for-covid-19
Stebbing, J., Phelan, A., Griffin, I., Tucker, C., Oechsle, O., Smith, D., & Richardson, P. (2020). COVID-19: combining antiviral and anti-inflammatory treatments. The Lancet Infectious Diseases, 20(4), 400-402. https://doi.org/10.1016/S1473-3099(20)30132-8
Morse, J. S.; Lalonde, T.; Xu, S.; Liu, W. R. (2020). Learning from the Past: Possible Urgent Prevention and Treatment Options for Severe Acute Respiratory Infections Caused by 2019‐nCoV. ChemBioChem 21 (5), 730–738. https://doi.org/10.1002/cbic.202000047.
Chang, G.-G. Quaternary Structure of the SARS Coronavirus Main Protease. In Molecular Biology of the SARS-Coronavirus; Lal, S. K., Ed.; Springer Berlin Heidelberg: Berlin, Heidelberg, 2010; pp 115–128. https://doi.org/10.1007/978-3-642-03683-5_8.