Targeting viral condensates could be a valuable strategy for developing fast-acting, specifically targeted drugs with a potential broad spectrum of activity against pathogenic viruses, including respiratory syncytial virus (RSV), by targeting proteins critical for condensate formation, according to an international collaborative study.
"A broad spectrum of activity whereby common condensate proteins within a viral family are found and targeted is a potential benefit of targeting condensates, since the potentially faster onset of action could be advantageous in acute respiratory infection," said study leader Ralf Altmeyer.
Condensate-targeting drugs may also enable the pharmacological modulation of previously undruggable targets, say the study authors, the first to demonstrate the feasibility of this approach in RSV-infected mice.
However, "although a broad activity broad spectrum is a potential [benefit] of condensate antivirals, this was not demonstrated in the present study," said Altmeyer, a professor in the Helmholtz International Lab for Anti-Infectives at Shandong University in Qingdao, China, when the study was conducted.
Indeed, "the activity [of our condensate-targeting candidate molecule] against RSV replication was very specific in terms of the molecular target and its inhibition profile against other viruses," said Altmeyer, who is currently affiliated with the School for Public Health at the University of Hong Kong.
"'Condensate' is short for 'liquid-liquid phase separation (LLPS) biomolecular condensate,' also known as a viral condensate," he explained.
In RSV, "the RSV cytoplasmic inclusion body (IB), or IB condensate, is an LLPS biomolecular condensate, which shares common features of condensates, namely liquidity, having no membrane, and formation from proteins and nucleic acid."
Biomolecular condensates have recently been demonstrated in physiological processes including embryonic development, cellular response to stress and pathological protein aggregation in neurodegenerative diseases, as well as cancer.
RSV is a major cause of respiratory illness in young children, the elderly, and the immune-compromised worldwide, but treatment options are limited and mainly supportive.
The use of ribavirin, the only FDA-approved antiviral treatment for severe RSV infections, is controversial, "due to safety and safety concerns, hence the need for better treatments," Altmeyer told BioWorld Science."
Consequently, multiple targets are currently under investigation for the development of safer and more effective RSV therapies.
In infected cells, RSV induces the formation of cytoplasmic IBs, in which nucleoprotein (N), phosphoprotein (P), the transcription factor M2-1 and viral genomic RNA are concentrated.
IBs have recently been described as being 'viral factories' for viral RNA synthesis, with their morphology suggesting that they are condensates formed by LLPS.
A recent study showed that N and P could drive formation of pseudo-IB condensates via LLPS in vitro, but these are not functional, as they neither harbor RNA synthesis nor reflect IB complexity in RSV-infected cells, which have multiple compartments.
Similar in size and phase organization to the nucleolus condensate, RSV IBs are multiphasic and have an IB-associated granule subcompartment comprising newly synthesized viral mRNA and M2-1.
Condensates have therefore emerged as an important subcellular organizing principle, but a key question in antiviral drug development concerns whether these condensates are druggable.
Theoretically, a drug that dissolved or hardened condensates should prevent viral replication, although neither mechanism has yet been reported.
In their new study reported in the July 7, 2021, edition of Nature, the authors showed that cyclopamine (CPM), and its analogue A3E, inhibited RSV replication by disorganizing and hardening IB condensates.
"CPM was originally identified as a specific nanomolar 'hit compound' during RSV phenotypic screening performed by CombinatoRx Pte. Ltd. in Singapore in 2008," said Altmeyer.
A naturally occurring plant-derived steroidal alkaloid, CPM is a teratogen that interrupts the hedgehog intracellular signaling pathway, resulting in fatal birth defects.
Therefore "we did not pursue it as drug candidate for obvious reasons, but in subsequent years I studied CPM's mechanism of action (MoA) at several academic institutions in China and in collaboration with our partners in France, culminating in the present study," Altmeyer explained.
"Under microscopy using fluorescence recovery after photobleaching (FRAP), CPM and A3E were shown to inhibit RSV replication by disorganizing and hardening IB condensates, which were seen to lose their mobility, becoming hard and dysfunctional," he said.
The researchers then demonstrated that the actions of CPM and its A3E analogue were blocked by a point mutation in the RSV transcription factor M2-1, establishing target validation.
"It is genetic evidence that M2-1 is a key component in the MoA of CPM and A3E and that there are no off-target effects that could explain that inhibition," explained Altmeyer.
Importantly, A3E and cyclopamine were further demonstrated to inhibit RSV in the lungs of infected mice, "by measuring viral replication in the lung," he said.
The two molecules therefore acted as condensate-targeting drug-like small molecules with in vivo activity.
Regarding safety, "as previously reported in a hitherto unpublished preliminary reproductive toxicology study, we did not see any teratogenic effects with A3E at the doses studies used for efficacy studies."
Together, these data suggest that condensate-hardening drugs may enable pharmacological modulation of previously undruggable targets in viral replication and possibly in cancer.
Looking ahead, said Altmeyer, "we will further optimize A3E and seek to identify new agents with which to induce condensate hardening through similar mechanisms, to which end we are forming a biotech company to develop clinical candidates."