Ipomoeassin F is a potent natural
cytotoxin that inhibits growth
of many tumor cell lines with single-digit nanomolar potency. However,
its biological and pharmacological properties have remained largely
unexplored. Building upon our earlier achievements in total synthesis
and medicinal chemistry, we used chemical proteomics to identify Sec61α
(protein transport protein Sec61 subunit alpha isoform 1), the pore-forming
subunit of the Sec61 protein translocon, as a direct binding partner
of ipomoeassin F in living cells. The interaction is specific and
strong enough to survive lysis conditions, enabling a biotin analogue
of ipomoeassin F to pull down Sec61α from live cells, yet it
is also reversible, as judged by several experiments including fluorescent
streptavidin staining, delayed competition in affinity pulldown, and
inhibition of TNF biogenesis after washout. Sec61α forms the
central subunit of the ER protein translocation complex, and the binding
of ipomoeassin F results in a substantial, yet selective, inhibition
of protein translocation in vitro and a broad ranging
inhibition of protein secretion in live cells. Lastly, the unique
resistance profile demonstrated by specific amino acid single-point
mutations in Sec61α provides compelling evidence that Sec61α
is the primary molecular target of ipomoeassin F and strongly suggests
that the binding of this natural product to Sec61α is distinctive.
Therefore, ipomoeassin F represents the first plant-derived, carbohydrate-based
member of a novel structural class that offers new opportunities to
explore Sec61α function and to further investigate its potential
as a therapeutic target for drug discovery.
Morbilliviruses, such as measles virus (MeV) and canine distemper virus (CDV), are highly infectious members of the paramyxovirus family. MeV is responsible for major morbidity and mortality in non-vaccinated populations. ERDRP-0519, a pan-morbillivirus small molecule inhibitor for the treatment of measles, targets the morbillivirus RNA-dependent RNA-polymerase (RdRP) complex and displayed unparalleled oral efficacy against lethal infection of ferrets with CDV, an established surrogate model for human measles. Resistance profiling identified the L subunit of the RdRP, which harbors all enzymatic activity of the polymerase complex, as the molecular target of inhibition. Here, we examined binding characteristics, physical docking site, and the molecular mechanism of action of ERDRP-0519 through label-free biolayer interferometry, photoaffinity cross-linking, and in vitro RdRP assays using purified MeV RdRP complexes and synthetic templates. Results demonstrate that unlike all other mononegavirus small molecule inhibitors identified to date, ERDRP-0519 inhibits all phosphodiester bond formation in both de novo initiation of RNA synthesis at the promoter and RNA elongation by a committed polymerase complex. Photocrosslinking and resistance profiling-informed ligand docking revealed that this unprecedented mechanism of action of ERDRP-0519 is due to simultaneous engagement of the L protein polyribonucleotidyl transferase (PRNTase)-like domain and the flexible intrusion loop by the compound, pharmacologically locking the polymerase in pre-initiation conformation. This study informs selection of ERDRP-0519 as clinical candidate for measles therapy and identifies a previously unrecognized druggable site in mononegavirus L polymerase proteins that can silence all synthesis of viral RNA.
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