Pharmacological inhibition of the HEG1-KRIT1 interaction

The Krüppel-like Factors 4 and 2 (KLF4/2) are transcription factors and master regulators of endothelial cells (ECs) phenotype and homeostasis. KLF4/2 are important blood-flow-responsive genes within ECs that differentially regulate the expression of factors that confer anti-inflammatory, antithrombotic, and antiproliferative effects in ECs. Genetic inactivation of endothelial HEG1 (Heart of glass) or KRIT1 (Krev interaction trapped protein 1) lead to upregulation of KLF4/2 expression levels. Furthermore, increased expression of thrombomodulin (THBD) and suppression of thrombospondin (THBS1) was ascribed to elevation of KLF4/2 as a result of loss of endothelial KRIT1. Here, we developed a high-throughput screening assay to identify inhibitors of the HEG1-KRIT1 interaction and identified, HEG1-KRIT1 inhibitor 1 (HKi1), as a promising hit inhibitor. The crystal structure of HKi1 bound to the KRIT1 FERM domain confirmed the primary screening results and ultimately led to the identification of a fragment-like inhibitor (HKi3), which occupies the HEG1 pocket producing comparable activity. These findings suggest that these inhibitors block the interaction by competing with the HEG1 for binding to KRIT1 FERM domain. Moreover, our results demonstrate that HKi3 upregulates KLF4/2 gene expression in two types of human ECs. These results reveal that acute pharmacological inhibition of the HEG1-KRIT1 interaction rapidly induces expression of KLF4/2 and their important transcriptional targets thrombomodulin and thrombospondin. Overall design: HUVECs were cultured in M199 medium (Sigma) supplemented with 10% FBS, 4 U/ml heparin, 3 ng/ml ß-EC growth factor (sigma), 4 U/ml heparin, and 100 units/ml penicillin-streptomycin. HUVECs were treated with a HEG-KRIT1 inhibitor (HKi3) or vehicle control (DMSO) for 24 hours. Four biological replicates were generated for vehicle control conditions and five replicates were generated for HKi3 treatment conditions. Following treatment, total mRNA was extracted, processed, and sequenced on a Illumina HiSeq4000.