Structural and mechanistic insights into the inhibition of respiratory syncytial virus polymerase by a non-nucleoside inhibitor

Abstract

The respiratory syncytial virus polymerase complex, consisting of the polymerase (L) and phosphoprotein (P), catalyzes nucleotide polymerization, cap addition, and cap methylation via the RNA dependent RNA polymerase, capping, and Methyltransferase domains on L. Several nucleoside and non-nucleoside inhibitors have been reported to inhibit this polymerase complex, but the structural details of the exact inhibitor-polymerase interactions have been lacking. Here, we report a non-nucleoside inhibitor JNJ-8003 with sub-nanomolar inhibition potency in both antiviral and polymerase assays. Our 2.9 Å resolution cryo-EM structure revealed that JNJ-8003 binds to an induced-fit pocket on the capping domain, with multiple interactions consistent with its tight binding and resistance mutation profile. The minigenome and gel-based de novo RNA synthesis and primer extension assays demonstrated that JNJ-8003 inhibited nucleotide polymerization at the early stages of RNA transcription and replication. Our results support that JNJ-8003 binding modulates a functional interplay between the capping and RdRp domains, and this molecular insight could accelerate the design of broad-spectrum antiviral drugs.

Structural and functional analysis of the Nipah virus polymerase complex

“…We did not observe density for the L CD, CTD, and MTase domains, despite using the full-length L protein in our preparations as confirmed by LC-MS/MS and based on the mass of assembled L–P 4 complexes detected by mass photometry (Figures 1B and S2). Therefore, like in structures of full-length L proteins of respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) ( Pneumoviridae ), the three L C-terminal globular domains in NiV L are probably too flexible relative to the RdRp-CAP polymerase core to be visualized 20–25 . For the domains that could be resolved (RdRp and CAP), alignment between NiV L and other nsNSV L based on the Cα ranged from 1.7 to 5.0 Å (Supplementary Table 2), indicating structural conservation, consistent with the generally similar transcription and replication mechanisms of nsNSVs.…”

Small Molecule Drugs Targeting Viral Polymerases

“…As of now, the RCSB PDB hosts six detailed cryo-EM structures which offer a detailed visualization of the overall architecture and organization of the RSV polymerase complex (PDB IDs: 6UEN [132]; 6PZK [134]; 8FPI [135]; 8FU3 [136]; 8SNX; 8SNY [137]). Specifically, these structures elucidate how the L and P proteins interact, their relative positioning, and the arrangement of domains within the polymerase assembly.…”

Recent Progress toward the Discovery of Small Molecules as Novel Anti-Respiratory Syncytial Virus Agents

“…Intriguingly, 49 ’s binding induced structural changes, revealing the plasticity of this pocket, a feature absent in the Apo form of the RSV L polymerase. Notably, the binding pocket of 49 differed from that of 31 , which retained its anti-RSV activity even against the RSV strain carrying the C1388G mutation . One year later, researchers at Janssen, in collaboration with various universities, reported the identification of the pyrazolopyrimidine analogue 50 (JNJ-7184, Figure ) as an allosteric NNI of RSV L polymerase .…”

“…108,129 In 2023, Yu and colleagues at Janssen (Johnson & Johnson) disclosed the discovery of a novel polymerase inhibitor following an extensive medicinal chemistry campaign that involved the synthesis and evaluation of approximately 5000 analogues. 130,131 Among these, compound 49 (JNJ-8003, Figure 16), characterized by a cinnoline scaffold, emerged as the most promising derivative. Notably, 49 demonstrated an exceptionally low EC 50 of 0.78 nM in a reporter assay using RSV A2 strain in HeLa cells (CC 50 = 27 μM).…”

“…Notably, the binding pocket of 49 differed from that of 31, which retained its anti-RSV activity even against the RSV strain carrying the C1388G mutation. 131 One year later, researchers at Janssen, in collaboration with various universities, reported the identification of the pyrazolopyrimidine analogue 50 (JNJ-7184, Figure 16) as an allosteric NNI of RSV L polymerase. 132 This identification was made following a HTS of an undisclosed library tested on HeLa cells infected with the RSV A2 reporter strain.…”