Wei Tang

Lonafarnib, an oral antiviral that targets the fusion glycoprotein of respiratory syncytial virus (RSV), has demonstrated efficacy in vitro and in vivo . However, because the RSV has evolved to become resistant to other fusion inhibitors, there is a concern that the same could occur for lonafarnib. Here, we identified resistance to lonafarnib in the RSV A2 strain and a recent clinical isolate, RSV ON1, via in vitro selection at scale. Cell‒cell fusion and recombinant live RSV analysis confirmed that the mutations at K394, K399, and T400 of the cysteine-rich region of the fusion protein mediate high-level resistance. Lonafarnib resistance mutations also confer cross-resistance to other fusion inhibitors of clinical interest. All-atom molecular dynamics simulations revealed that these resistance mutations confer reduced stability to the fusion protein, thereby diminishing its binding affinity with lonafarnib. To address this vulnerability proactively and increase the barrier to resistance development, we designed the first potent proteolysis-targeting chimera (PROTAC) fusion protein degrader, compound 0179841, which uses lonafarnib and cereblon as ligands. This PROTAC effectively inhibited RSV replication. Collectively, our findings indicate that RSV develops resistance to lonafarnib in the cysteine-rich region of the fusion protein. This work sheds light on the mechanisms by which RSV evolves resistance to lonafarnib and provides a foundation for the rational design of antivirals aimed at preventing resistance. IMPORTANCE: Respiratory syncytial virus (RSV) infection poses a substantial public health challenge. Resistance to several potent fusion inhibitors, which are currently in various stages of clinical development, can readily emerge. Through a drug repurposing screen, we identified lonafarnib as an RSV fusion inhibitor; however, concerns exist regarding the potential development of resistance. Here, large-scale in vitro selection experiments revealed specific mutations within the highly conserved cysteine-rich region of the fusion (F) protein that confer high-level lonafarnib resistance across diverse RSV strains. These resistance mutations also confer cross-resistance to other clinical-stage fusion inhibitors. Mechanistic investigations demonstrated that these mutations reduce F protein stability, thereby diminishing the binding affinity of lonafarnib. As a proof of concept for an alternative antiviral strategy, we rationally designed the first potent proteolysis-targeting chimera (PROTAC) F protein degrader, compound 0179841, by utilizing lonafarnib and cereblon ligands. This novel antiviral agent effectively inhibits RSV infection by inducing degradation of the F protein. This work elucidates the molecular basis of RSV resistance to lonafarnib and establishes a strategy for developing next-generation antivirals aimed at preempting resistance.

Respiratory syncytial virus (RSV) entry into host cells is facilitated by viral fusion, wherein the metastable RSV fusion (F) protein undergoes a conformational change from a prefusion state to a highly stable postfusion structure. The prefusion F elicits a more robust human antibody response than its postfusion F and is a primary target for RSV vaccine development. However, the inherent instability of the prefusion F trimer and its low protein expression level in host cells are a significant challenge for developing a high-potency RSV vaccine. Here, we report that the introduction of four hydrophobic residue substitutions in the RSV F protein resulted in a highly stable prefusion F trimer (pre-F-IFLP). This engineered variant exhibits enhanced expression and stability compared to DS-Cav1, with improved thermal stability, increased resistance to acid and base, and extended storage life. Furthermore, pre-F-IFLP induced neutralizing antibody responses 72-fold higher than those elicited by DS-Cav1 following a second booster immunization and fully protected mice against RSV infection. IMPORTANCE: In this study, we demonstrate that introducing four hydrophobic residue substitutions into the RSV F protein leads to the generation of a highly stable prefusion F trimer (pre-F-IFLP) with improved expression levels in cultured cells and superior stability compared to DS-Cav1, the first-generation prefusion F-stabilized RSV vaccine. Furthermore, pre-F-IFLP induced significantly higher neutralizing antibody responses than DS-Cav1 following both the first and second booster immunizations and conferred complete protection against RSV infection in a mouse model. These findings present an alternative approach for stabilizing the trimeric prefusion F protein, enhancing its expression, and significantly improving its protective efficacy for the prevention of RSV infection in vivo .

Herpes simplex virus type 1 (HSV-1) causes keratoconjunctivitis and encephalitis, with nucleoside analogues such as acyclovir (ACV) serving as standard treatments. However, rising drug resistance underscores the need for agents with novel mechanisms. In this study, phytochemical investigation led to the obtainment of several phloroglucinol derivatives from Rhodomyrtus tomentosa. Subsequent phenotypic screening demonstrated compound 6 as the most promising HSV-1 inhibitor. To facilitate lead optimization, we synthesized 44 analogues of 6 to elucidate the structure-activity relationships, and 24 analogues exhibited comparable potency to 6 against both HSV-1 and ACV-resistant viruses. Preliminary mechanistic studies revealed that 6 can inhibit viral replication by blocking host cell factors involved in serine 5-phosphorylated RNA polymerase expression. Furthermore, 6 also showed efficacy in vivo against HSV-1 infection via topical administration. These findings highlight a new type of anti-HSV-1 inhibitors with a mechanism distinct from that of ACV, offering new avenues for anti-HSV-1 drug development.

Infection with respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract disease in young children and older people. Despite intensive efforts over the past few decades, no direct-acting small-molecule agents against RSV are available. Most small-molecule candidates targeting the RSV fusion (F) protein pose a considerable risk of inducing drug-resistant mutations. Here, we explored the in vitro and in vivo virological properties of the K394R variant, a cross-resistant mutant capable of evading multiple RSV fusion inhibitors. Our results demonstrated that the K394R variant is highly fusogenic in vitro and more pathogenic than the parental strain in vivo . The small molecule (2 E ,2′ E )- N , N ′-((1 R ,2 S ,3 S )−3-hydroxycyclohexane-1,2-diyl)bis(3-(2-bromo-4-fluorophenyl) acrylamide) (CL-A3-7), a structurally optimized compound derived from a natural caffeoylquinic acid derivative, substantially reduced in vitro and in vivo infections of both wild-type RSV and the K394R variant. Mechanistically, CL-A3-7 significantly inhibited virus–cell fusion during RSV entry by blocking the interaction between the viral F protein and the cellular insulin-like growth factor 1 receptor (IGF1R). Collectively, these results indicate severe disease risks caused by the K394R variant and reveal a new anti-RSV mechanism to overcome K394R-associated resistance. IMPORTANCE: Respiratory syncytial virus (RSV) infection is a major public health concern, and many small-molecule candidates targeting the viral fusion (F) protein are associated with a considerable risk of inducing drug-resistant mutations. This study investigated virological features of the K394R variant, a mutant strain conferring resistance to multiple RSV fusion inhibitors. Our results demonstrated that the K394R variant is highly fusogenic in cell cultures and more pathogenic than the parental strain in mice. The small-molecule inhibitor CL-A3-7 substantially reduced in vitro and in vivo infections of both wild-type RSV and the K394R variant by blocking the interaction of viral F protein with its cellular receptor, showing a new mechanism of action for small-molecules to inhibit RSV infection and overcome K394R-associated resistance.

Four new meroterpenoids (1–4) with novel skeletons were isolated from Xanthostemon chrysanthus. Compound 1 is the first phloroglucinol-based meroterpenoid with a 16-membered macrocyclic ring and shows strong antiviral activity against RSV and HSV-1.

A synthetic strategy based on biogenetic building blocks for the collective and divergent biomimetic synthesis of cleistoperlones A−F, a cinnamoylphloroglucinol collection discovered from Cleistocalyx operculatus, has been developed. These syntheses proceeded successfully in only six to seven steps starting from commercially available 1,3,5‐benzenetriol and involving oxidative activation of stable biogenetic building blocks as a crucial step. Key features of the syntheses include a unique Michael addition/ketalization/1,6‐addition/enol‐keto tautomerism cascade reaction for the construction of the dihydropyrano[3,2‐d]xanthene tetracyclic core of cleistoperlones A and B, and a rare inverse‐electron‐demand hetero‐Diels–Alder cycloaddition for the establishment of benzopyran ring in cleistoperlones D−F. Moreover, cleistoperlone A exhibited significant antiviral activity against acyclovir‐resistant strains of herpes simplex virus type 1 (HSV‐1/Blue and HSV‐1/153).

Human infection with highly pathogenic H5N1 influenza virus causes severe respiratory diseases. Currently, the drugs against H5N1 are limited to virus-targeted inhibitors. However, drug resistance caused by these inhibitors is becoming a serious threat to global public health. An alternative strategy to reduce the resistance risk is to develop antiviral drugs targeting host cell proteins. In this study, we demonstrated that cytochrome c oxidase subunit 4 isoform 1 (COX41) of host cell plays an important role in H5N1 infection. Overexpression of COX41 promoted viral replication, which was inhibited by silencing or knockout the expression of COX41 in the host cell. The ribonucleoproteins (RNPs) of H5N1 were retained in the cell nucleus after knockout cellular COX41. Strikingly, inhibition of cellular COX41 by lycorine, a small-molecule compound isolated from Amaryllidaceae plants, reduced the levels of COX41-induced ROS and phosphorylation of extracellular signal-regulated kinase (ERK) in cells, thus resulting in the blockage of nuclear export of vRNP and inhibition of viral replication. In H5N1-infected mice that were treated with lycorine, we observed a reduction of viral titers and inhibition of pathological changes in the lung and trachea tissues. Importantly, no resistant virus was generated after culturing the virus with the continuous treatment of lycorine. Collectively, these findings suggest that COX41 is a positive regulator of H5N1 replication and might serve as an alternative target for anti-influenza drug development.