Discovery and Optimization of a Series of Benzofuran Selective ERAP1 Inhibitors: Biochemical and In Silico Studies.

ERAP1 is a key aminopeptidase involved in peptide trimming before major histocompatibility complex (MHC) presentation. A single nucleotide polymorphism (SNP) in the ERAP1 gene can lead to impaired trimming activity and affect ERAP1 function. ERAP1 genetic variations have been linked to an increased susceptibility to cancer and autoimmune disease. Here, we report the discovery of novel ERAP1 inhibitors using a high throughput screening approach. Due to ERAP1 broad substrate specificity, the hit finding strategy included testing inhibitors with a range of biochemical assays. Based on the hit potency, selectivity, and in vitro absorption, distribution, metabolism, excretion, and toxicity, the benzofuran series was selected. Fifteen derivatives were designed and synthesized, the compound potency was improved to the nanomolar range, and the structure-activity relationship supported by modeling studies.

The Dinoflagellate Toxin 20-Methyl Spirolide-G Potently Blocks Skeletal Muscle and Neuronal Nicotinic Acetylcholine Receptors.

The cyclic imine toxin 20-methyl spirolide G (20-meSPX-G), produced by the toxigenic dinoflagellate Alexandrium ostenfeldii/Alexandrium peruvianum, has been previously reported to contaminate shellfish in various European coastal locations, as revealed by mouse toxicity bioassay. The aim of the present study was to determine its toxicological profile and its molecular target selectivity. 20-meSPX-G blocked nerve-evoked isometric contractions in isolated mouse neuromuscular preparations, while it had no action on contractions elicited by direct electrical stimulation, and reduced reversibly nerve-evoked compound muscle action potential amplitudes in anesthetized mice. Voltage-clamp recordings in Xenopus oocytes revealed that 20-meSPX-G potently inhibited currents evoked by ACh on Torpedo muscle-type and human α7 nicotinic acetylcholine receptors (nAChR), whereas lower potency was observed in human α4ß2 nAChR. Competition-binding assays showed that 20-meSPX-G fully displaced [³H]epibatidine binding to HEK-293 cells expressing the human α3ß2 (Ki = 0.040 nM), whereas a 90-fold lower affinity was detected in human α4ß2 nAChR. The spirolide displaced [(125)I]α-bungarotoxin binding to Torpedo membranes (Ki = 0.028 nM) and in HEK-293 cells expressing chick chimeric α7-5HT3 nAChR (Ki = 0.11 nM). In conclusion, this is the first study to demonstrate that 20-meSPX-G is a potent antagonist of nAChRs, and its subtype selectivity is discussed on the basis of molecular docking models.

Marine Macrocyclic Imines, Pinnatoxins A and G: Structural Determinants and Functional Properties to Distinguish Neuronal α7 from Muscle α1(2)ßγδ nAChRs.

Pinnatoxins are macrocyclic imine phycotoxins associated with algal blooms and shellfish toxicity. Functional analysis of pinnatoxin A and pinnatoxin G by binding and voltage-clamp electrophysiology on membrane-embedded neuronal α7, α4ß2, α3ß2, and muscle-type α12ßγδ nicotinic acetylcholine receptors (nAChRs) reveals high-affinity binding and potent antagonism for the α7 and α12ßγδ subtypes. The toxins also bind to the nAChR surrogate, acetylcholine-binding protein (AChBP), with low Kd values reflecting slow dissociation. Crystal structures of pinnatoxin-AChBP complexes (1.9-2.2 Å resolution) show the multiple anchoring points of the hydrophobic portion, the cyclic imine, and the substituted bis-spiroketal and cyclohexene ring systems of the pinnatoxins that dictate tight binding between the opposing loops C and F at the receptor subunit interface, as observed for the 13-desmethyl-spirolide C and gymnodimine A congeners. Uniquely, however, the bulky bridged EF-ketal ring specific to the pinnatoxins extends radially from the interfacial-binding pocket to interact with the sequence-variable loop F and govern nAChR subtype selectivity and central neurotoxicity.