Ecdysteroid ligand–receptor selectivity – exploring trends to design orthogonal gene switches

Abstract

A set of thirty-two natural and ten semisynthetic ecdysteroids was assayed in murine 3T3 cells across ten different ecdysteroid receptor (EcR) ligandbinding domains derived from nine arthropod species in an engineered gene switch format. Among the ecdysteroids tested, the most biologically widespread ecdysteroid, 20-hydroxyecdysone (20E), was moderately and consistently potent across the tested EcRs. The most potent ligand-receptor combination (EC 50 = 0.3 nm) was ponasterone A (PoA) actuating the Nephotettix cincticeps EcR switch. The most robust ligand-receptor combination, as measured by potency and efficacy, was PoA actuating either the Bombyx mori EcR or a 'VY' (E274V ⁄ V390I ⁄ Y410E) mutant of Choristoneura fumiferana EcR. Parallel ecdysteroid structure-activity relationships were observed across species; addition of hydroxyl groups at positions 2, 3, 14, 20 and 22 incrementally enhanced potency, whereas hydroxylation at position 25 retarded potency. Nevertheless, several outlier ligand-EcR combinations, such as cyasterone actuating the VY C. fumiferana EcR mutant and canescensterone activating Bemisia argentifolii EcR, exhibited an inversion of relative potency, and therefore lend themselves to construction of orthogonal duplex gene switches. The potency inversion between these two ligand-receptor pairs can be accounted for by steroid-tail contact residues Tyr411 and Met502 in VY C. fumiferana EcR corresponding to two threonines in B. argentifolii EcR. Another potency inversion was also observed with cyasterone operating on the VY mutant of C. fumiferana EcR and polypodine B activating Aedes aegypti EcR. The ecdysteroid-EcR dataset, generated in a non-natural system, nevertheless invites conjecture regarding relative ecdysteroid potencies, plant species distribution of certain phytoecdysteroids, and the role of phytoecdysteroids as chemodefense against relevant insect herbivores.

Semi‐Synthetic Ecdysteroids as Gene‐Switch Actuators: Synthesis, Structure–Activity Relationships, and Prospective ADME Properties

“…Potency is retained or improved by differ- [68] Consistent with an H-bond acceptor assignment is also the observation that steroids lacking a 22-OH group altogether are substantially depleted in potency. [33] Available crystal structures of PoA bound to Tribolium castaneum EcR [31] and 20E bound to HvEcR [32] indicate water-mediated H-bridges between 22-OH and a conserved Asp residue.…”

The Karlson Lecture. Phytoecdysteroids: What use are they?

“…Of these, only SAR of ecdysteroid nuclear receptors has been exploited significantly (reviewed in Dinan, 2003;Dinan and Hormann, 2005), and has been used to develop pharmacophore hypotheses for the interaction of the ligand with the ligand-binding domain of the receptor (Dinan et al, 1999), to investigate the mode of interaction between ligand and receptor (Lapenna et al, 2009), and to explore the ligand specificities of ecdysteroid receptors from different species (Lapenna et al, 2008). The latter has also been investigated by X-ray crystallography after cocrystallisation of a phytoecdysteroid with the LBD (Billas and Moras, 2005).…”

Absolute Configuration and Docking Study of Canescensterone, a Potent Phytoecdysteroid, with Non‐Lepidopteran Ecdysteroid Receptor Selectivity

“…In a multi-receptor gene-switch screening employing 10 different EcR ligandbinding domains (LBDs) from nine arthropod species, this phytoecdysteroid showed an approximately constant inwww.eurjoc.orgcrease in responsiveness towards the six non-lepidopteran EcRs over the four lepidopteran EcRs studied, both in terms of potency (EC 50 ) and efficacy [RMFI (maximum fold induction relative to a gene-switch activator standard)]. [11] In particular, the highest responsiveness was obtained with the EcR from the homopteran Bemisia argentifolii (Ba; silverleaf whitefly) for which EC 50 = 0.2 µ and RMFI = 4.881 (cf. EC 50 Ͼ 10 µ and RMFI ≈ 0.001 with lepidopteran EcRs).…”

“…[18,32] The complete stereochemical assignment of canescensterone allowed a docking study to be undertaken aimed at unravelling the molecular basis of canescensterone selectivity. BaEcR and VY-CfEcR are suitable representative proteins for this task, not only because of the availability of homogeneous activity data with both these receptors, [11] but also because of the existence of X-ray crystallography data for identical or closely related EcR LBDs complexed to an ecdysteroid analogue, that is, the EcR LBDs from the hemipteran Bemisia tabaci (Bt; sweet potato whitefly) complexed with PoA, [19] which is identical to the BaEcR LBD, and the lepidopteran Heliotis virescens (Hv; tobacco budworm) complexed with PoA [20b] and 20E, [20a] which differs for two residues from the VY-CfEcR LBD. The binding pose of the ligand is essentially identical in all the ecdysteroid-bound EcR crystal structures deciphered to date.…”

“…In contrast, canescensterone shows a remarkable selectivity for non-lepidopteran EcRs, with an approximately 50-fold increase in potency and almost 5000-fold increase in efficacy over lepidopteran EcRs (moths and butterflies) when measured against a set of 10 EcRs representing nine arthropod species in a common gene-switch format. [11] Canescensterone is a rare phytoecdysteroid isolated from the bark of Vitex canescens, a tree endemic to Thailand. [12] This compound possesses an unusual ecdysteroid structure insofar as it lacks the typical hydroxy group at the 22-position, which usually enhances activity, whereas it bears a pyrrole-2-carboxylate group attached to C-24, a moiety not yet found in any other natural ecdysteroid.…”