A Pan-Respiratory Antiviral Chemotype Targeting a Host Multi-Protein Complex

Abstract

We present a novel small molecule antiviral chemotype that was identified by an unconventional cell-free protein synthesis and assembly-based phenotypic screen for modulation of viral capsid assembly. Activity of PAV-431, a representative compound from the series, has been validated against infectious virus in multiple cell culture models for all six families of viruses causing most respiratory disease in humans. In animals this chemotype has been demonstrated efficacious for Porcine Epidemic Diarrhea Virus (a coronavirus) and Respiratory Syncytial Virus (a paramyxovirus). PAV-431 is shown to bind to the protein 14-3-3, a known allosteric modulator. However, it only appears to target the small subset of 14-3-3 which is present in a dynamic multi-protein complex whose components include proteins implicated in viral lifecycles and in innate immunity. The composition of this target multi-protein complex appears to be modified upon viral infection and largely restored by PAV-431 treatment. Our findings suggest a new paradigm for understanding, and drugging, the host-virus interface, which leads to a new clinical therapeutic strategy for treatment of respiratory viral disease.

SciScore for 10.1101/2021.01.17.426875: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	not detected.
Randomization	In the 150 pig trial done, a number of different litters of newborn pigs were all infected and then randomized to control (vehicle) and compound.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	RSV cotton rat study: Twenty-four female cotton rats, ~5 weeks of age, were obtained from Envigo (formerly Harlan), ear-tagged for identification purposes and allowed to acclimate for > 1 week prior to study start.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Selected antibodies were purchased from Bethyl Laboratories, Inc (rabbit polyclonal affinity purified antibody to VCP/p97, …

SciScore for 10.1101/2021.01.17.426875: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	not detected.
Randomization	In the 150 pig trial done, a number of different litters of newborn pigs were all infected and then randomized to control (vehicle) and compound.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	RSV cotton rat study: Twenty-four female cotton rats, ~5 weeks of age, were obtained from Envigo (formerly Harlan), ear-tagged for identification purposes and allowed to acclimate for > 1 week prior to study start.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Selected antibodies were purchased from Bethyl Laboratories, Inc (rabbit polyclonal affinity purified antibody to VCP/p97, catalogue number A300-588A-T) and Abcam (mouse monoclonal antibody to human p62, catalogue number ab56416).	human p62 suggested: (Abcam Cat# ab56416, RRID:AB_945626)
Western blotting: For western blotting, SDS/PAGE gels were transferred in Towbin buffer to polyvinylidene fluoride membrane, blocked in 1% BSA, incubated for 1 hour at room temperature in a 1:1,000 dilution of 100 μg/mL affinity-purified primary IgG, washed three times in PBS with 0.1% Tween-20, and incubated for 1 hour in a 1:5,000 dilution of secondary anti-rabbit antibody coupled to alkaline phosphatase.	anti-rabbit suggested: None alkaline phosphatase. suggested: None
Experimental Models: Cell Lines
Sentences	Resources
FLUV antiviral assay: Inhibition of FLUV replication was assayed using infection of MDCK.2 cells.	MDCK.2 suggested: BCRJ Cat# 0170, RRID:CVCL_B034)
SARS-CoV-2 assay: SARS-CoV-2 (2019-nCoV/USA-WA1/2020; MN985325.1) was received from BEI resources and propagated in Vero clone E6, Vero E6, CRL-1586.	Vero suggested: ATCC Cat# CRL-1586, RRID:CVCL_0574) Vero E6 suggested: None
SIV and BoCoV assays: MDCK Cells (100 μL) at a density of 1×106 – 1×107/mL in MEM plus 5% - 10% FBS were seeded in 96-well plates.	MDCK suggested: None
MRC-5 cells infected with FLUV A/WSN/33 or BoCoV and for each infected sample a sample that was treated 1 hour after infection with compound PAV-431 for 24 hours.	MRC-5 suggested: None
Software and Algorithms
Sentences	Resources
These were all done in duplicate and the calculations were performed using Prism8 from GraphPad.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

Of course, every scientific method has its strengths and its limitations, the point being to take advantage of the former and recognize the latter. CFPSA is just one new and specialized experimental tool. Second, the confounding nature of both the energy-dependence (and therefore transience) of the MPC target and the biochemical heterogeneity of its individual protein components explains why these targets have been heretofore missed. With regards to energy-dependence, we are not aware of previous demonstration or use of energy-dependent affinity chromatography. If, as our data suggests, independent energy-dependent steps are involved in the formation/binding of an MPC to an affinity ligand (in this case the drug), and for release from the affinity ligand, the discovery of this new class of targets may have impact far beyond the therapeutics of respiratory viruses described here. Moreover the transience of such targets suggests relevance for a new understanding of the molecular basis for homeostasis with myriad therapeutic implications. With regards to protein heterogeneity, if only 5 percent or less of a given protein represents the subset found in an MPC target of interest (as demonstrated here for VCP/p97), then efforts focusing on that gene product by methods that don’t allow subset discrimination may fail to detect the relevance of that subset for the target of interest. It should be noted that a burgeoning literature on the multifunctionality of proteins1,50, including i...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

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No protocol registration statement was detected.

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A Pan-Respiratory Antiviral Chemotype Targeting a Host Multi-Protein Complex

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