Synthesis, Identification, and Structure–Activity Relationship Analysis of GATA4 and NKX2-5 Protein–Protein Interaction Modulators

Abstract

Transcription factors GATA4 and NKX2-5 directly interact and synergistically activate several cardiac genes and stretch-induced cardiomyocyte hypertrophy. Previously, we identified phenylisoxazole carboxamide 1 as a hit compound, which inhibited the GATA4–NKX2-5 transcriptional synergy. Here, the chemical space around the molecular structure of 1 was explored by synthesizing and characterizing 220 derivatives and structurally related compounds. In addition to the synergistic transcriptional activation, selected compounds were evaluated for their effects on transcriptional activities of GATA4 and NKX2-5 individually as well as potential cytotoxicity. The structure–activity relationship (SAR) analysis revealed that the aromatic isoxazole substituent in the southern part regulates the inhibition of GATA4–NKX2-5 transcriptional synergy. Moreover, inhibition of GATA4 transcriptional activity correlated with the reduced cell viability. In summary, comprehensive SAR analysis accompanied by data analysis successfully identified potent and selective inhibitors of GATA4–NKX2-5 transcriptional synergy and revealed structural features important for it.

GATA-targeted compounds modulate cardiac subtype cell differentiation in dual reporter stem cell line

“…In order to test for the effects of GATA4-targeted compounds on differentiation programs of cardiomyocyte subtypes (atrial vs ventricular), a differentiation assay based on the expression of markers of ventricular (Myl2-eGFP, venGFP) and atrial (SMyHC3-TdTomato, atrRFP) cardiomyocytes was used [ 43 ]. As compounds affect GATA4/NKX2-5 protein-protein interactions [ 38 , 39 ], and it is unknown to what extent these protein-protein interactions have temporal characteristics during the differentiation process, compounds were tested during a broad treatment window (D2–D10) representing both mesodermal commitment and activation of differentiation markers (Fig. 1 a).…”

“…Flow cytometry was performed on a BD Accuri C6 or BD LSRFortessa flow cytometer. Synthesis of compounds used in the present study was performed in the Division of Pharmaceutical Chemistry at the University of Helsinki, Pharmatory (Oulu, Finland), Chembridge (San Diego, USA), and Maybridge (Leicestershire, UK) as described [ 38 , 39 ]. All-trans retinoic acid (ATRA) and (+)-JQ1 were purchased from a commercial provider (Sigma).…”

“…Moreover, we have previously identified GATA4 and NKX2-5 as master regulators of stretch-induced hypertrophic responses in differentiated cardiomyocytes [ 36 ] and reported the structural basis for the GATA4/NKX2-5 interaction [ 37 ]. The observed nuclear receptor-like structure of the GATA4/NKX2-5 complex provides an opportunity for small molecule interference, and we subsequently reported a novel family of compounds targeting the GATA4/NKX2-5 interaction that inhibited synergistic transcription from reporter genes possessing NKX2-5 binding sites [ 38 , 39 ]. Furthermore, we showed that the hit compound 3i-1000 inhibited cardiomyocyte hypertrophy in vitro and improved left ventricular ejection fraction/structural remodeling after myocardial infarction and other cardiac injuries in vivo [ 39 – 41 ].…”

Smad4 regulates the nuclear translocation of Nkx2-5 in cardiac differentiation

“…Moreover, GATA4 reportedly binds to an upstream region of the Nkx2-5 transcriptional start site and controls Nkx2-5 www.nature.com/scientificreports/ expression in cooperation with Smad1/4 22 . Some studies have also reported an interaction between GATA4 and Nkx2-5 35,36 . Thus, after the Smad4-Nkx2-5 complex translocates into the nucleus, a complex containing Smad4, Nkx2-5, and GATA4 may allow for the sustained expression of Nkx2-5, thereby acting as a maintenance mechanism for mature cardiomyocytes.…”

GATA4-targeted compound exhibits cardioprotective actions against doxorubicin-induced toxicity in vitro and in vivo: establishment of a chronic cardiotoxicity model using human iPSC-derived cardiomyocytes

“…Recently, we have reported the identification of small molecules that either inhibit or enhance the GATA4-NKX2-5 transcriptional synergy (Jumppanen et al 2019;Välimäki et al 2017). The most potent inhibitor of GATA4-NKX2-5 interaction, 3i-1000, had no influence on the baseline GATA4 proteins levels in NRVMs, whereas the phenylephrine-induced elevation in GATA4 Ser-105 phosphorylation was significantly inhibited by 3i-1000 (Kinnunen et al 2018).…”