In vivo structure-activity relationship study of dorsomorphin analogues identifies selective VEGF and BMP inhibitors

Abstract

The therapeutic potential of small molecule signaling inhibitors is often limited by off-target effects. Recently, in a screen for compounds that perturb the zebrafish embryonic dorsoventral axis, we identified dorsomorphin, the first selective inhibitor of bone morphogenetic protein (BMP) signaling. Here we show that dorsomorphin has significant "off-target" effects against the VEGF (vascular endothelial growth factor) type-2 receptor (Flk1/KDR) and disrupts zebrafish angiogenesis. Since both BMP and VEGF signals are known to be involved in vascular development, we sought to determine whether dorsomorphin's antiangiogenic effects are due to its impact on the BMP or VEGF signals through the development of analogues that target BMP but not VEGF signaling and vice versa. In a structure-activity relationship (SAR) study of dorsomorphin analogues based primarily on their effects on live zebrafish embryos, we identified highly selective and potent BMP inhibitors as well as selective VEGF inhibitors. One of the BMP inhibitors, DMH1, which exclusively targets the BMP but not the VEGF pathway, dorsalized the embryonic axis without disrupting the angiogenic process, demonstrating that BMP signaling was not involved in the angiogenic process. This is one of the first full-scale SAR studies performed in vertebrates and demonstrates the potential of zebrafish as an attractive complementary platform for drug development that incorporates an assessment of in vivo bioactivity and selectivity in the context of a living organism.

Figure 1. Dorsomorphin inhibits both BMP and VEGF signaling, and the pyrazolo[1,5-a]pyrimidine backbone of DM for derivatization

(a) Structure of dorsomorphin (DM). (b) 2 μM DM dorsalized zebrafish embryos when administered at 3 hours post fertilization (hpf), and 10 μM DM blocked intersomitic vessel (ISV) formation when administered at 12-hpf. Above, bright-field images treated embryos. Below, green fluorescence images of the Tg(Fli:EGFP)y1 transgenic embryos, which express GFP in the vasculature. Control embryos treated with DMSO are on left, and DM-treated embryos are on right. (c) DM suppressed VEGF-dependent Flk1 phosphorylation in a dose-dependent manner in bovine arterial endothelial cells (BAECs). (d) The pyrazolo[1,5-a]pyrimidine backbone of DM for derivatization involving modifications at the 3- and 6- positions (red circles).

Figure 2. Schema of zebrafish-based structure-activity relationship (SAR) study of DM analogs

To assess each analog’s effect on dorsoventral (DV) axis, the wild-type embryos were exposed to the compound (at concentration from 0.01 μM to 50 μM) starting at 3-hpf. To assess each analog’s effect on angiogenesis, the Tg(Fli:EGFP)y1 embryos were exposed to each compound (from 0.01 μM to 50 μM) starting at 12-hpf. After 48 hrs, the compound-treated embryos were manually scored for dorsalization of the DV axis (BMPR-I inhibition), vascular defects (VEGFR-2 inhibition), and nonspecific toxicity or defects. The compounds which caused dorsalization or blocked angiogenesis were considered for the subsequent round of analog synthesis and testing.

Figure 3. Chemical Structures, IUPAC Nomenclatures and LC/MS analyses of DMH1, DMH2, DMH3 and DMH4

DMH1: 4-(6-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline; >98% @ 214 nM, Rt = 2.48 min, m/z = 381.2 [M + H]. DMH2: 4-(2-(4-(3-(quinolin-4-yl)pyrazolo[1,5-a]pyrimidin-6-yl)phenoxy)ethyl)morpholine; >98% @ 214 nM, Rt = 0.72 min, m/z = 452.2 [M + H] DMH3: N,N-dimethyl-3-(4-(3-(quinolin-4-yl)pyrazolo[1,5-a]pyrimidin-6-yl)phenoxy)propan-1-amine; >98% @ 214 nM, Rt = 0.76 min, m/z = 424.3 [M + H] DMH4: 4-(2-(4-(3-phenylpyrazolo[1,5-a]pyrimidin-6-yl)phenoxy)ethyl)morpholine; >98% @ 214 nM, R_T = 0.89 min., m/z = 401.2 [M + H].

Figure 4. DMH1 is a potent and selective inhibitor of BMP signaling

(a) DMH1 (0.2 μM) caused severe dorsalization when the embryos were treated from 3-hpf (above), but it (100 μM) had no effect on ISV formation when the embryos were treated from 12-hpf (below, green fluorescence marks vascular endothelium). (b) DMH1 inhibited BMP signaling in a dose dependent manner in BMP-responsive luciferase reporter assay. RLU (relative luciferase units). (c) DMH1 blocked BMP4-induced Smad 1/5/8 phosphorylation in HEK293 cells. In contrast, DMH1 had no effect on (c) BMP4-induced p38 MAPK phosphorylation, and on (d) Activin A-induced Smad2 phosphorylation in HEK293 cells. (e) DMH1 had no effect on VEGF-induced Flk1 phosphorylation in BAECs.

Figure 5. DMH4 is a potent and selective inhibitor of VEGF signaling

(a) DMH4 disrupted both ISV and SIV formation at 1 μM. Fluorescent vascular images of DMSO-treated embryo are shown on the left of DMH4-treated embryos. (b) DMH4 had no effect on dorsoventral axis at 50 μM when treated from 3-hpf. (c) DMH4 (1 μM) blocked VEGF-induced tubular network formation in HUVECs. (d) DMH4 did not show significant inhibition of BMP signaling in a BMP-responsive luciferase reporter assay, but (e, f) it blocked VEGF-induced phosphorylation of Flk1 and AKT in BAECs.