Adenosine 5'-monophosphate activated protein kinase (AMPK) has emerged as a major potential target for novel antidiabetic drugs. We studied the structure of 2-chloro-5-((Z)-((E)-5-((5-(4,5-dimethyl-2-nitrophenyl)furan-2-yl)methylene)-4-oxothiazolidin-2-ylidene)amino)benzoic acid (PT-1), which attenuates the autoinhibition of the enzyme AMPK, for the design and synthesis of different benzothiazoles with potential antidiabetic activity. We synthesized several structurally related benzothiazole derivatives that increased the rate of glucose uptake in L6 myotubes in an AMPK-dependent manner. One compound, 2-(benzo[d]thiazol-2-ylmethylthio)-6-ethoxybenzo[d]thiazole (34), augmented the rate of glucose uptake up to 2.5-fold compared with vehicle-treated cells and up to 1.1-fold compared to PT-1. Concomitantly, it elevated the abundance of GLUT4 in the plasma membrane of the myotubes and activated AMPK. Subcutaneous administration of 34 to hyperglycemic Kuo Kondo rats carrying the Ay-yellow obese gene (KKAy) mice lowered blood glucose levels toward the normoglycemic range. In accord with its activity, compound 34 showed a high fit value to a pharmacophore model derived from the PT-1.

G protein-coupled receptors (GPCRs) are cell-surface receptors that respond to various stimuli to induce signalling pathways across cell membranes. Recent progress has yielded atomic structures of key intermediates1,2 and roles for lipids in signalling3,4. However, capturing signalling events of a wild-type receptor in real time, across a native membrane to its downstream effectors, has remained elusive. Here we probe the archetypal class A GPCR, rhodopsin, directly from fragments of native disc membranes using mass spectrometry. We monitor real-time photoconversion of dark-adapted rhodopsin to opsin, delineating retinal isomerization and hydrolysis steps, and further showing that the reaction is significantly slower in its native membrane than in detergent micelles. Considering the lipids ejected with rhodopsin, we demonstrate that opsin can be regenerated in membranes through photoisomerized retinal–lipid conjugates, and we provide evidence for increased association of rhodopsin with unsaturated long-chain phosphatidylcholine during signalling. Capturing the secondary steps of the signalling cascade, we monitor light activation of transducin (Gt) through loss of GDP to generate an intermediate apo-trimeric G protein, and observe Gαt•GTP subunits interacting with PDE6 to hydrolyse cyclic GMP. We also show how rhodopsin-targeting compounds either stimulate or dampen signalling through rhodopsin–opsin and transducin signalling pathways. Our results not only reveal the effect of native lipids on rhodopsin signalling and regeneration but also enable us to propose a paradigm for GPCR drug discovery in native membrane environments.

Degeneration of retinal photoreceptor cells can arise from environmental and/or genetic causes. Since photoreceptor cells, the retinal pigment epithelium (RPE), neurons, and glial cells of the retina are intimately associated, all cell types eventually are affected by retinal degenerative diseases. Such diseases often originate either in rod and/or cone photoreceptor cells or the RPE. Of these, cone cells located in the central retina are especially important for daily human activity. Here we describe the protection of cone cells by a combination therapy consisting of the G protein-coupled receptor modulators metoprolol, tamsulosin, and bromocriptine. These drugs were tested in mice, a preclinical model for retinal degeneration. The specificity of these drugs was determined with an essentially complete panel of human G protein-coupled receptors. Significantly, the combination of metoprolol, tamsulosin, and bromocriptine had no deleterious effects on electroretinographic responses of wild-type mice. Moreover, putative G protein-coupled receptor targets of these drugs were shown to be expressed in human and mouse eyes by RNA sequencing and quantitative polymerase chain reaction. Liquid chromatography together with mass spectrometry using validated internal standards confirmed that metoprolol, tamsulosin, and bromocriptine individually or together penetrate the eye after either intraperitoneal delivery or oral gavage. Collectively, these findings support human trials with combined therapy composed of lower doses of metoprolol, tamsulosin, and bromocriptine designed to safely impede retinal degeneration associated with certain genetic diseases (e.g., Stargardt disease). The same low-dose combination also could protect the retina against diseases with complex or unknown etiologies such as age-related macular degeneration.

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