We have identified chemical probes that simultaneously inhibit cancer cell progression and an immune checkpoint. Using the computational Site Identification by Ligand Competitive Saturation (SILCS) technology, structural biology and cell-based assays, we identify small molecules that directly and selectively bind to the RNA Recognition Motif (RRM) of hnRNP A18, a regulator of protein translation in cancer cells. hnRNP A18 recognizes a specific RNA signature motif in the 3′UTR of transcripts associated with cancer cell progression (Trx, VEGF, RPA) and, as shown here, a tumor immune checkpoint (CTLA-4). Post-transcriptional regulation of immune checkpoints is a potential therapeutic strategy that remains to be exploited. The probes target hnRNP A18 RRM in vitro and in cells as evaluated by cellular target engagement. As single agents, the probes specifically disrupt hnRNP A18–RNA interactions, downregulate Trx and CTLA-4 protein levels and inhibit proliferation of several cancer cell lines without affecting the viability of normal epithelial cells. These first-in-class chemical probes will greatly facilitate the elucidation of the underexplored biological function of RNA Binding Proteins (RBPs) in cancer cells, including their effects on proliferation and immune checkpoint activation.
The heterogeneous ribonucleoprotein A18 (hnRNP A18) is upregulated in hypoxic regions of various solid tumors and promotes tumor growth via the coordination of mRNA transcripts associated with pro-survival genes. Thus, hnRNP A18 represents an important therapeutic target in tumor cells. Presented here is the first X-ray crystal structure to be reported for the RNA-recognition motif of hnRNP A18. By comparing this structure with those of homologous RNA-binding proteins (i.e. hnRNP A1), three residues on one face of an antiparallel β-sheet (Arg48, Phe50 and Phe52) and one residue in an unstructured loop (Arg41) were identified as likely to be involved in protein-nucleic acid interactions. This structure helps to serve as a foundation for biophysical studies of this RNA-binding protein and structure-based drug-design efforts for targeting hnRNP A18 in cancer, such as malignant melanoma, where hnRNP A18 levels are elevated and contribute to disease progression.
Engineering polyketide synthases (PKS) to produce new metabolites requires an understanding of catalytic points of failure during substrate processing. Growing evidence indicates the thioesterase (TE) domain as a significant bottleneck within engineered PKS systems. We created a series of hybrid PKS modules bearing exchanged TE domains from heterologous pathways and challenged them with both native and non‐native polyketide substrates. Reactions pairing wildtype PKS modules with non‐native substrates primarily resulted in poor conversions to anticipated macrolactones. Likewise, product formation with native substrates and hybrid PKS modules bearing non‐cognate TE domains was severely reduced. In contrast, non‐native substrates were converted by most hybrid modules containing a substrate compatible TE, directly implicating this domain as the major catalytic gatekeeper and highlighting its value as a target for protein engineering to improve analog production in PKS pathways.
Engineering polyketide synthases (PKS) to produce new metabolites requires an understanding of catalytic points of failure during substrate processing. Growing evidence indicates the thioesterase (TE) domain as a significant bottleneck within engineered PKS systems. We created a series of hybrid PKS modules bearing exchanged TE domains from heterologous pathways and challenged them with both native and non‐native polyketide substrates. Reactions pairing wildtype PKS modules with non‐native substrates primarily resulted in poor conversions to anticipated macrolactones. Likewise, product formation with native substrates and hybrid PKS modules bearing non‐cognate TE domains was severely reduced. In contrast, non‐native substrates were converted by most hybrid modules containing a substrate compatible TE, directly implicating this domain as the major catalytic gatekeeper and highlighting its value as a target for protein engineering to improve analog production in PKS pathways.
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