Discovery of Novel Small Molecule Inhibitors of Multiple Influenza Strains in Cell Culture

Petsch, Benjamin; Hurt, Clarence R.; Freeman, Beverly; Zirdum, Elisabeth; Ganesh, Anupama; Schörg, Alexandra; Kitaygorodskyy, Anatoliy; Marwidi, Yoko; Ducoudret, Olivier; Kelleher, CC; Hansen, Wendy F.; Lingappa, Vishwanath R.; Essrich, Christian; Stitz, Lothar

doi:10.1016/j.antiviral.2010.02.398

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“…Antibodies recognizing the FLUV NP were used to establish an ELISA-style plate assay for FLUV capsid assembly (Figure 2A) 16 as previously achieved for rabies virus, rift valley fever virus (RVFV) and HIV 8,17,18 . A portion of a 150,000 drug-like small molecule library was screened and a number of hits identified from completely different chemical classes (Tanimoto similarity coefficient < 40%), three of which were advanced by medicinal chemistry analog synthesis (Figure 2B).…”

Section: Resultsmentioning

confidence: 99%

“…Much of this prior work involved CFPSA systems programmed with mRNA encoding viral capsid proteins. A moderate throughput phenotypic drug screen involving CFPSA of influenza (FLUV) encoded proteins was developed (Petsch et al, 2010), analogous to what has also been done for rabies (Lingappa et al, 2013), HIV (Copeland et al, 2010; Reed et al, 2021), and other viruses (Broce et al, 2016). This screen is carried out in cellular extracts rather than in living cells, with formation of multimeric capsid protein complexes as a quantifiable, functional endpoint (Harrell, E.K.T.…”

Section: Resultsmentioning

confidence: 99%

“…A cell-free protein synthesis and assembly (CFPSA) based phenotypic screen was established for influenza (FLUV) analogous to what has been done for rabies, HIV, and other viruses (13,19,40,41). Unlike conventional phenotypic screens, this screen was carried out in cellular extracts rather than in living cells.…”

Section: Resultsmentioning

confidence: 99%

“…Assembly was assessed by co-migration of multiple newly synthesized FLUV gene products in the final sucrose step gradient (ssg) peak. The timedependent high molecular weight peak in fraction 10/11 was subsequently analyzed by iodixanol buoyant density gradients (idxg), and showed multiple viral gene products comigrating in the expected buoyant density range of authentic FLUV particles 16 . Expression of the capsid proteins of other respiratory viral families, including SARS-CoV-2, a member of the family Coronaviridae (Figure 1B), and RSV of the family Paramyxoviridae (data not shown), also displayed similar assembly pathways, albeit with features distinctive to each viral family.…”

Section: Probing Viral Assembly With An Orthogonal Host-targeted Viral Protein Biogenesis Screenmentioning

confidence: 99%

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

Selvarajah¹,

Lingappa²,

Michon³

et al. 2021

Preprint

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Antiviral compounds displaying remarkable features have been identified by an unconventional drug screen and advanced through animal validation. Efficacy is observed against the six viral families causing most human respiratory viral disease, irrespective of strain, including both influenza (FLUV) and SARS-CoV-2, with cell culture EC50 at or below 100 nM. Survival benefit is demonstrated in pigs against another member of family Coronaviridae, porcine epidemic diarrhea virus (PEDV), and shown equally effective in mild and severe disease. Respiratory syncytial virus (RSV) titer is reduced by drug treatment in cotton rats. A substantial barrier to viral resistance is demonstrated for FLUV. Drug resin affinity chromatography (DRAC) reveals a novel drug target: a multi-protein complex (MPC) formed transiently, in an energy-dependent fashion, and composed of host proteins implicated in both viral lifecycles and manipulation of innate immunity. The protein composition of this host MPC is modified upon viral infection, with increase or decrease of some proteins and appearance or complete loss of others. Valosin-containing protein, also known as Transitional endoplasmatic reticulum ATPase (VCP/p97), is present in the target MPC of uninfected cells and significantly increased in both FLUV and CoV infection. SQSTM1/p62, a key regulator of the autophagy pathway of innate immunity whose dysfunction is implicated in cytokine storm, is i) found in the target MPC from uninfected cells, ii) diminished in DRAC eluates by infection, and iii) restored by drug treatment of infected cells. 14-3-3 is one of likely several proteins that comprise the drug-binding site. Advanced compounds with improved pharmacokinetic (PK) properties and lung exposure are approaching criteria for a Target Product Profile. We propose these novel drug targets to comprise a previously unappreciated molecular basis for homeostasis that is modified by viruses to allow exploitation for viral propagation and is restored by treatment with the therapeutic compounds presented. This discovery has transformative implications for treatment of respiratory viral-related disease, applicable to everything from seasonal FLUV to COVID-19 and future novel respiratory viruses, due to the pan-family nature of drug activity and barrier to resistance development.

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