Frank, A. O. | Search bioRxiv

bioRxiv2019

The Structural Basis of Indisulam-Mediated Recruitment of RBM39 to the DCAF15-DDB1-DDA1 E3 Ligase Complex

The anti-cancer agent Indisulam inhibits cell proliferation by causing degradation of RBM39, an essential mRNA splicing factor. Indisulam promotes an interaction between RBM39 and the DCAF15 E3 ligase substrate receptor leading to RBM39 ubiquitination and proteasome-mediated degradation. To delineate the precise mechanism by which Indisulam mediates DCAF15-RBM39 interaction, we solved the DCAF15-DDB1-DDA1-Indisulam-RBM39(RRM2) complex structure to 2.3 [A]. DCAF15 has a novel topology which embraces the RBM39(RRM2) domain largely via nonpolar interactions, and Indisulam binds between DCAF15 and RBM39(RRM2) and coordinates additional interactions between the two proteins. Studies with RBM39 point mutants and Indisulam analogs validated the structural model and defined the RBM39 alpha-helical degron motif. The degron is found only in RBM23 and RBM39 and only these proteins were detectably downregulated in Indisulam-treated HCT116 cells. This work further explains how Indisulam induces RBM39 degradation and defines the challenge of harnessing DCAF15 to degrade novel targets.

biophysics↗

bioRxiv2019

A VP2/3-derived peptide exhibits potent antiviral activity against BK and JC polyomaviruses by targeting a novel VP1 binding site

In pursuit of effective therapeutics for human polyomaviruses, we identified a peptide derived from the BK polyomavirus (BKV) minor structural proteins VP2/3 that is a potent inhibitor of BKV infection with no observable cellular toxicity. The thirteen amino acid peptide binds to major structural protein VP1 in a new location within the pore with a low nanomolar KD. Alanine scanning of the peptide identified three key residues, substitution of each of which results in [~]1000-fold loss of affinity with a concomitant reduction in antiviral activity. NMR spectroscopy and an X-ray structurally-guided model demonstrate specific binding of the peptide to the pore of the VP1 pentamer that constitutes the BKV capsid. Cell-based assays with the peptide demonstrate nanomolar inhibition of BKV infection and suggest that the peptide likely blocks the viral entry pathway between endocytosis and escape from the host cell ER. The peptide motif is highly conserved among the polyomavirus clade, and homologous peptides exhibit similar binding properties for JC polyomavirus and inhibit infection with similar potency to BKV in a model cell line. Substitutions within VP1 or VP2/3 residues involved in VP1-peptide interaction negatively impact viral infectivity, potentially indicating the peptide-binding site within the VP1 pore is relevant for VP1-VP2/3 interactions. The inhibitory potential of the peptide-binding site first reported here may present a novel target for development of new anti-polyomavirus therapies. In summary, we present the first anti-polyomavirus inhibitor that acts via a novel mechanism of action by specifically targeting the pore of VP1.

microbiology↗