We
report the design, synthesis, and biological evaluation of some
potent small-molecule neuropilin-1 (NRP1) antagonists. NRP1 is implicated
in the immune response to tumors, particularly in Treg cell fragility,
required for PD1 checkpoint blockade. The design of these compounds
was based on a previously identified compound EG00229. The design
of these molecules was informed and supported by X-ray crystal structures.
Compound 1 (EG01377) was identified as having properties
suitable for further investigation. Compound 1 was then
tested in several in vitro assays and was shown to have antiangiogenic,
antimigratory, and antitumor effects. Remarkably, 1 was
shown to be selective for NRP1 over the closely related protein NRP2.
In purified Nrp1+, FoxP3+, and CD25+ populations of Tregs from mice, 1 was able to block
a glioma-conditioned medium-induced increase in TGFβ production.
This comprehensive characterization of a small-molecule NRP1 antagonist
provides the basis for future in vivo studies.
Neuropilin-1 (NRP1) is emerging as an important molecule in immune signaling where it has been shown to modulate the actions of TGF-β1 in macrophages and regulatory T cells. The development of cost-effective and reliable assays for NRP1 binding is therefore important. We synthesized three new NRP1 small molecule fluorophores and examined their performance as fluorescent polarization probes. One molecule DS108 exhibited favorable binding and fluorescent characteristics and allowed us to establish a simple assay suitable for medium to high throughput screening of small molecules.
K E Y W O R D Sfluorescence polarization, neuropilin-1, surface plasmon resonance
BackgroundHeme oxygenase (HO) catalyzes NADPH-dependent degradation of heme to liberate iron, carbon monoxide and biliverdin. The interaction between HO and cytochrome P450 reductase (CPR), an electron donor, is essential for HO activity. HO-1 is a stress-inducible isoform whereas HO-2 is constitutively expressed. HO-1 induction is commonly seen in cancers and impacts disease progression, supporting the possibility of targeting HO-1 for cancer therapy.MethodsWe employed a cell-based bioluminescence resonance energy transfer assay to screen compounds with ability to inhibit HO-1/CPR interaction. The effect of the identified compound on HO-1/CPR interaction was confirmed by pull down assay. Moreover, the anti-tumorigenic activity of the identified compound on HO-1-enhanced tumor growth and migration was assessed by trypan blue exclusion method and wound healing assay.ResultsDanthron was identified as an effective small molecule able to interfere with the interaction between HO-1 and CPR but not HO-2 and CPR. Additional experiments with structural analogues of danthron revealed that the positions of hydroxyl moieties significantly affected the potency of inhibition on HO-1/CPR interaction. Pull-down assay confirmed that danthron inhibited the interaction of CPR with HO-1 but not HO-2. Danthron suppressed growth and migration of HeLa cells with stable HO-1 overexpression but not mock cells. In contrast, anthrarufin, a structural analog with no ability to interfere HO-1/CPR interaction, exhibited no significant effect on HO-1-overexpressing HeLa cells.ConclusionsThese findings demonstrate that danthron is an isoform-specific inhibitor for HO-1/CPR interaction and may serve as a lead compound for novel anticancer drug.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.