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Highly Potent Host-Specific Small-Molecule Inhibitor of Paramyxovirus and Pneumovirus Replication with High Resistance Barrier - PubMed

  • ️Fri Jan 01 2021

Highly Potent Host-Specific Small-Molecule Inhibitor of Paramyxovirus and Pneumovirus Replication with High Resistance Barrier

Neeta Shrestha et al. mBio. 2021.

Abstract

Multiple enveloped RNA viruses of the family Paramyxoviridae and Pneumoviridae, like measles virus (MeV), Nipah virus (NiV), canine distemper virus (CDV), or respiratory syncytial virus (RSV), are of high clinical relevance. Each year a huge number of lives are lost as a result of these viral infections. Worldwide, MeV infection alone is responsible for over a hundred thousand deaths each year despite available vaccine. Therefore, there is an urgent need for treatment options to counteract these viral infections. The development of antiviral drugs in general stands as a huge challenge due to the rapid emergence of viral escape mutants. Here, we disclose the discovery of a small-molecule antiviral, compound 1 (ZHAWOC9045), active against several pneumo-/paramyxoviruses, including MeV, NiV, CDV, RSV, and parainfluenza virus type 5 (PIV-5). A series of mechanistic characterizations revealed that compound 1 targets a host factor which is indispensable for viral genome replication. Drug resistance profiling against a paramyxovirus model (CDV) demonstrated no detectable adaptation despite prolonged time of investigation, thereby mitigating the rapid emergence of escape variants. Furthermore, a thorough structure-activity relationship analysis of compound 1 led to the invention of 100-times-more potent-derivatives, e.g., compound 2 (ZHAWOC21026). Collectively, we present in this study an attractive host-directed pneumoviral/paramyxoviral replication inhibitor with potential therapeutic application. IMPORTANCE Measles virus, respiratory syncytial virus, canine distemper virus, and Nipah virus are some of the clinically significant RNA viruses that threaten substantial number of lives each year. Limited to no availability of treatment options for these viral infections makes it arduous to handle the outbreaks. This highlights the major importance of developing antivirals to fight not only ongoing infections but also potential future epidemics. Most of the discovered antivirals, in clinical trials currently, are virus targeted, which consequently poses the challenge of rapid emergence of escape variants. Here, we present compound 1 (ZHAWOC9045), discovered to target viral replication in a host-dependent manner, thereby exhibiting broad-spectrum activity against several members of the family Pneumo-/Paramyxoviridae. The inability of viruses to mutate against the inhibitor mitigated the critical issue of generation of escape variants. Importantly, compound 1 was successfully optimized to a highly potent variant, compound 2 (ZHAWOC21026), with a promising profile for pharmacological intervention.

Keywords: high resistance barrier; host-directed; inhibitors; paramyxovirus; pneumovirus; replication.

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Figures

FIG 1
FIG 1

Discovery of a novel small-molecule antiviral. (A) Structure of compound 1 (F2205-0189), compound 3 (3G), ERDRP-0519 (16), and JMN3-003 (19). (B) Assessment of the compounds’ inhibitory impact on virus-induced syncytium formation. Microscopic images of cells infected with OPneon in the presence of the compounds. Scale bars, 2,000 μm. Pictures were captured with a Cytation 5 imaging multimode reader (BioTek). Note that due to automatic settings, discrepancy in background intensity measurement is visible in the stitched images. (C) IC50 measurement of compounds against the attenuated OP-CDV strain. (D) IC50 measurement of compounds against the wild-type A75/17-CDV. (E) Measurement of the cytotoxic effect of the inhibitors. Ninety-five percent confidence intervals are shown in parentheses. Relative luminescence values were normalized for values obtained in the presence of DMSO control and represent means from three independent experiments. (F) Impact of compound 1 on cell cycle progression of treated cells. Vero cells were incubated in the absence or presence of increasing concentration of compound 1 for 42 h and analyzed using flow cytometry. The values show the means ± SD from three independent experiments. Dunnett’s multiple-comparison test was applied after two-way analysis of variance (ANOVA) (*, P < 0.05). (G) Assessment of viral proliferation after incubation with the compound. Vero cells expressing canine SLAM (cSLAM) were infected with wild-type A75/17-CDV (A75neon/nLucP) at an MOI of 0.01 in the presence of increasing concentrations of the compound. After 30 h, infected cells were frozen and thawed (twice), and viruses in the lysates were harvested and titrated in Vero-cSLAM cells.

FIG 2
FIG 2

General synthesis routes for the optimization of compound 1. We used two main strategies to synthesize most of the new inhibitors. In route I, we first built the ether moiety and coupled the aminothiazole moiety in the last step. This allows a convenient variation of the thiazole moiety. In route II, we first coupled this thiazole moiety followed by the introduction of the phenol building block to vary the other side of the scaffold. (a) NaOH in water at 100°C for 45 min (49). (b) N,N-Diisopropylethylamine (DIPEA) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU) in dimethylformamide (DMF) at 0°C to room temperature (RT) for 1 h. (c) Triethylamine (TEA) in dichloromethane (DCM) at 0°C to RT for 1 h. (d) Cs2CO3 and NaI in DMF at 60°C overnight.

FIG 3
FIG 3

Investigation of mode of action of compound 1. (A) IC50 measurement of compound 1 against the attenuated OP-CDV strain in either Vero cells or Vero cells expressing SLAM (cSLAM) or Nectin4 (cN4) receptors. (B) Investigation of virucidal effect of compound 1. As a positive control, compound 3 as a virucidal compound was added. (C) OP-CDV time-of-addition studies. Compound 3 (entry inhibitor) and ERDRP-0519 (replication inhibitor) were taken as references. Relative luminescence was measured after 48 h of initial infection. Relative luminescence values were normalized for values obtained in the presence of DMSO control and represent means ± SD from three independent experiments. (D) Plasmid-based minigenome luciferase assay to determine the bioactivity of CDV polymerase complex. Relative luminescence values were normalized for values obtained in the presence of DMSO control and represent means ± SD from three independent experiments. Statistical significance of differences was determined using one-way ANOVA followed by Dunnett’s multiple-comparison test (****, P < 0.0001; ns, not significant). (E) Antiviral activity of compound 1 is host cell species specific. IC50 values of compound 1 were measured against the attenuated OP-CDV strain in cell lines of different species origin. Ninety-five percent confidence intervals are shown in parentheses. (F) Intracellular localization of alkyne-tagged compound 1 (1-alk) assessed through a click reaction using azide-linked Alexa Fluor 488 in Vero and P114 cell lines. 20624 and 21320 were used as negative controls. Green shows Alexa Fluor 488, and blue shows DAPI (nuclear staining). (G) Structure of three alkyne-tagged compounds. (H) Compound 1 displays broad-spectrum activity. IC50 values of compound 1 were measured against various viruses in corresponding cell lines. NA, not applicable. IC50 values of compound 1 were measured against indicated viruses. Ninety-five percent confidence intervals are shown in parentheses.

FIG 4
FIG 4

Compound 1 impedes the rapid emergence of viral mutants. Wild-type A75/17-CDV was continuously adapted for 83 days in the presence of either ERDRP-0519 (virus directed) or compound 1 (host directed). Four independent adaptations were followed for compound 1 (green), whereas two independent adaptations were followed for ERDRP-0519 (brown).

FIG 5
FIG 5

Structure of the optimized inhibitor 2 (ZHAWOC21026) depicting the SAR conclusions. The black arrow visualizes the possible C=O···S interaction (50).

FIG 6
FIG 6

Generation of highly potent variants of compound 1. (A) IC50 values of one of the 3 best variants, compound 2, were measured against different members of families Paramyxoviridae and Pneumoviridae in corresponding cell lines. Ninety-five percent confidence intervals are shown in parentheses. (B) Impact of compound 2 on cell cycle progression of treated cells. Vero cells were incubated in the absence or presence of increasing concentrations of compound 2 for 42 h and analyzed using flow cytometry. The values indicate the means ± SD from three independent experiments. Dunnett’s multiple-comparison test was applied after two-way ANOVA (*, P < 0.05).

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