Importance of the Choice of a Recombinant System to Produce Large Amounts of Functional Membrane Protein hERG.

Human ether-a-gogo related gene (hERG) product is the membrane potassium channel Kv11.1, which is involved in the electrical activity of the heart. As such, it is a key player in the toxicity of many drug candidates. Therefore, having this protein at hand during earlier stages of drug discovery is important for preventing later toxicity. Furthermore, having a fair quantity of functional channels may help in the development of the necessary techniques for gaining insight in this channel structure. Thus, we performed a comparative study of methods for over-expressing a mutated but functional, hERG in different orthologous hosts, such as yeast, bacteria, insect and human cell lines. We also engineered the protein to test various constructs of a functional channel. We obtained a significant amount of a functional mutant channel from HEK cells that we thoroughly characterized. The present work paves the way for the expression of large amounts of this protein, with which protein crystallization or cryo-electronic microscopy will be attempted. This will be a way to gain information on the structure of the hERG active site and its modelization to obtain data on the pauses of various reference compounds from the pharmacopeia, as well as to gain information about the thermodynamics of the hERG/ligand relationship.

Melatonin MT1 and MT2 receptors display different molecular pharmacologies only in the G-protein coupled state.

BACKGROUND AND PURPOSE: Melatonin receptors have been extensively characterized regarding their affinity and pharmacology, mostly using 2-[(125)I]-melatonin as a radioligand. Although [(3)H]-melatonin has the advantage of corresponding to the endogenous ligand of the receptor, its binding has not been well described. EXPERIMENTAL APPROACH: We characterized [(3)H]-melatonin binding to the hMT1 and hMT2 receptors expressed in a range of cell lines and obtained new insights into the molecular pharmacology of melatonin receptors. KEY RESULTS: The binding of [(3)H]-melatonin to the hMT1 and hMT2 receptors displayed two sites on the saturation curves. These two binding sites were observed on cell membranes expressing recombinant receptors from various species as well as on whole cells. Furthermore, our GTPγS/NaCl results suggest that these sites on the saturation curves correspond to the G-protein coupled and uncoupled states of the receptors, whose pharmacology was extensively characterized. CONCLUSIONS AND IMPLICATIONS: hMT1 and hMT2 receptors spontaneously exist in two states when expressed in cell lines; these states can be probed by [(3)H]-melatonin binding. Overall, our results suggest that physiological regulation of the melatonin receptors may result from complex and subtle mechanisms, a small difference in affinity between the active and inactive states of the receptor, and spontaneous coupling to G-proteins.

Mutagenic analysis in a pure molecular system shows that thioredoxin-interacting protein residue Cys247 is necessary and sufficient for a mixed disulfide formation with thioredoxin.

The human thioredoxin (TRX)-interacting protein is found in multiple subcellular compartments and plays a major role in redox homeostasis, particularly in the context of metabolism (e.g., lipidemia and glycemia) and apoptosis. A molecular approach to the protein's modus operandi is still needed because some aspects of the TRX-interacting protein-mediated regulation of TRX are not clearly understood. To this end, His-tagged TRX-interacting proteins were over-expressed in Escherichia coli. Because the protein is expressed mainly in inclusion bodies, it was denatured in high concentrations of guanidium hydrochloride, centrifuged, and purified by Ni-NTA affinity chromatography. His-TRX-interacting protein was then refolded by dialysis and its restructuring monitored by circular dichroism spectrometry. This preparation resulted in the formation of a covalent complex with recombinant human TRX, demonstrating that association occurs without the intervention of other partner proteins. Multiple cysteine-to-serine mutants of TRX-interacting protein were produced and purified. These mutations were efficient in limiting the formation of disulfide-linked homo-oligomers in an oxidizing environment. The mutants were also used to gain functional insight into the formation of the TRX-interacting protein-TRX complexes. These complexes were able to form in the absence of internal disulfide bridges. A mutant with all but one cysteine changed to serine (Cys ²47) also showed an enhanced capacity to form complexes with TRX demonstrating, in a pure molecular system, that this particular cysteine is likely responsible for the disulfide bridge between TRX-interacting protein and TRX.

Molecular pharmacology of the mouse melatonin receptors MT1 and MT2.

The main melatonin receptors are two G-protein coupled receptors named MT(1) and MT(2). Having described the molecular pharmacology of the human versions of these receptors, we turned to two of the three species most useful in studying melatonin physiology: rat and sheep (a diurnal species used to understand the relationship between circadian rhythm and depression). We also employed previously used compounds to describe the mouse melatonin receptors; despite the early cloning of mouse receptors, few molecular pharmacology studies on these receptors exist. To our surprise, we detected no major differences between the data obtained from mice and those from other species.

Meganuclease-driven targeted integration in CHO-K1 cells for the fast generation of HTS-compatible cell-based assays.

The development of cell-based assays for high-throughput screening (HTS) approaches often requires the generation of stable transformant cell lines. However, these cell lines are essentially created by random integration of a gene of interest (GOI) with no control over the level and stability of gene expression. The authors developed a targeted integration system in Chinese hamster ovary (CHO) cells, called the cellular genome positioning system (cGPS), based on the stimulation of homologous gene targeting by meganucleases. Five different GOIs were knocked in at the same locus in cGPS CHO-K1 cells. Further characterization revealed that the cGPS CHO-K1 system is more rapid (2-week protocol), efficient (all selected clones expressed the GOI), reproducible (GOI expression level variation of 12%), and stable over time (no change in GOI expression after 23 weeks of culture) than classical random integration. Moreover, in all cGPS CHO-K1 targeted clones, the recombinant protein was biologically active and its properties similar to the endogenous protein. This fast and robust method opens the door for creating large collections of cell lines of better quality and expressing therapeutically relevant GOIs at physiological levels, thereby enhancing the potential scope of HTS.

S38151 [p-guanidinobenzoyl-[Des-Gly(10)]-MCH(7-17)] is a potent and selective antagonist at the MCH(1) receptor and has anti-feeding properties in vivo.

Structure-activity relationships studies have established the minimal sequence of melanin-concentrating hormone (MCH) that retains full agonist potency at the MCH(1), to be the dodecapeptide MCH(6-17). The alpha-amino function is not required for activity since arginine(6) can be replaced by p-guanidinobenzoyl, further improving activity. We report that the deletion of glycine in this short potent agonist (EC(50) 3.4nM) turns it into a potent and new MCH(1) antagonist (S38151, K(B) 4.3nM in the [(35)S]-GTPgammaS binding assay), which is selective versus MCH(2). A compared Ala-scan of the agonist and antagonist sequences reveals major differences in the residues that are mandatory for affinity, including arginine(11) and tyrosine(13) for the agonist and leucine(9) for the antagonist, whereas methionine(8) was necessary for both agonist and antagonist activities. A complete molecular study of the antagonist behavior is described in the present report, with a particular focus on the description of several analogues, attempting to find structure-activity relationships. Finally, S38151 antagonizes food intake when injected intra-cerebroventricularly in the rat. This is in agreement with the in vitro data and with our previous demonstration of a good correlation between in vitro and in vivo data on MCH(1) agonists.