Design of a small molecule that stimulates vascular endothelial growth factor A enabled by screening RNA fold–small molecule interactions

Abstract

Vascular endothelial growth factor A (VEGFA) stimulates angiogenesis in human endothelial cells, and increasing its expression is a potential treatment for heart failure. Here, we report the design of a small molecule (TGP-377) that specifically and potently enhances VEGFA expression by the targeting of a non-coding microRNA that regulates its expression. A selection-based screen, named two-dimensional combinatorial screening, revealed preferences in small-molecule chemotypes that bind RNA and preferences in the RNA motifs that bind small molecules. The screening program increased the dataset of known RNA motif–small molecule binding partners by 20-fold. Analysis of this dataset against the RNA-mediated pathways that regulate VEGFA defined that the microRNA-377 precursor, which represses Vegfa messenger RNA translation, is druggable in a selective manner. We designed TGP-377 to potently and specifically upregulate VEGFA in human umbilical vein endothelial cells. These studies illustrate the power of two-dimensional combinatorial screening to define molecular recognition events between ‘undruggable’ biomolecules and small molecules, and the ability of sequence-based design to deliver efficacious structure-specific compounds.

Frameworks for targeting RNA with small molecules

“…This work suggested that bioactive RNA-targeted ligands may occupy a focused corner of drug-like chemical space. These results were further supported by recent work from the Disney laboratory in collaboration with AstraZeneca (57). The researchers observed that although the RNA-binding hit compounds were structurally dissimilar to those in R-BIND (113), they still shared the identified physicochemical properties.…”

“…2 ). Using a technique termed two-dimensional combinatorial screening, the Disney laboratory constructed the proposed database, now known as Inforna, which they have used to target several RNAs with small molecules possessing activity in cell culture and/or animal models of cancer and neurological disorders ( 53 , 54 , 55 , 57 , 58 , 59 , 60 ). A key strategy in Disney's work has been that of modular assembly, where at least two binding moieties are linked together to interact with neighboring secondary structure motifs such as those found in precursor miRNAs and trinucleotide repeat RNAs ( Fig.…”

“…The idea that secondary structure motifs can serve as modules, or units, for targeting RNA has delivered several bioactive small molecules ( 53 , 54 , 55 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 ). These successes testify to the strength of this approach as a general RNA-targeting strategy.…”

Small molecule recognition of disease-relevant RNA structures

“…In addition to the design of homodimeric molecules, Inforna can be used to design heterodimeric compounds which bind avidly to miRNAs. 49 Vascular endothelial growth factor A (VEGFA) stimulates angiogenesis in human endothelial cells and is a sought after target in the treatment of heart failure. [50][51][52] MiR-377 regulates VEGFA expression, and repression of miR-377 by an antisense oligonucleotide has been shown to rescue VEGFA expression and stimulate angiogenesis.…”

“…2F and Table S1, ESI †). 49 Expression levels of miR-377 from human umbilical vein endothelial cells (HUVEC) treated with TGP-377 were knocked down with an IC 50 of B500 nM, 10-fold more potently than the lead small molecule monomer (Table S1, ESI †). Accumulation of pre-miR-377 was also observed, demonstrating TGP-377 acts through inhibition of Dicer processing and correspondingly rescues VEGFA expression.…”