Jean-Luc Butour

ORL1, a novel member of the opioid receptor family : cloning, functional expression and localization

Selective PCR amplification of human and mouse genomic DNAs with oligonucleotides encoding highly conserved regions of the δ‐opioid and somatostatin receptors generated a human DNA probe (hOP01, 761 bp) and its murine counterpart (mOP86, 447 bp). hOP01 was used to screen a cDNA library from human brainstem. A clone (named hORL1) was isolated, sequenced and found to encode a protein of 370 amino acids whose primary structure displays the seven putative membrane‐spanning domains of a G protein‐coupled membrane receptor. The hORL1 receptor is most closely related to opioid receptors not only on structural (sequence) but also on functional grounds: hORLl is 49–50% identical to the murine μ‐, δ‐ and κ‐opioid receptors and, in CHO‐K1 cells stably transfected with a pRc/CMV:hORLl construct, ORL1 mediates inhibition of adenylyl cyclase by etorphine, a ‘universal’ (nonselective) opiate agonist. Yet, hORLl appears not to be a typical opioid receptor. Neither is it a somatostatin or σ (N‐allylnormetazocine) receptor. mRNAs hybridizing with synthetic oligonucleotides complementary to mOP86 are present in many regions of the mouse brain and spinal cord, particularly in limbic (amygdala, hippocampus, septum, habenula,⋯) and hypothalamic structures. We conclude that the hORL1 receptor is a new member of the opioid receptor family with a potential role in modulating a number of brain functions, including instinctive behaviours and emotions.

Modifications of the DNA secondary structure upon platinum binding: a proposed model

Summary A number of physico-chemical techniques were used to study the interaction between a series of platinum compounds and DNA. Spectrofluorometry, ultraviolet spectroscopy (UV), circular dichroism (CD), melting curves, kinetics and electron microscopy (EM) were found powerful in the differentiation of the DNA secondary structure modifications upon platinum binding. Three types of alterations are clearly shown and can be correlated with the mode of platinum binding to DNA. Type I is typical of a cis attack, type II of a trans attack and type III, of a monofunctional platinum binding. In type III, no significant DNA modifications were noticed in UV, CD, fluorescence and EM. The different results obtained with cis and trans platinum compounds indicate a strong perturbation of the DNA structure upon bidentate platinum binding. These two types of compounds were found to shorten the DNA length as shown in EM. A correlation was found between the kinetics of the DNA-Platinum interaction for a series of cis-compounds and their activity on leukemia L1210 in mice. A model for the cis-Pt(NH3)2Cl2 interaction with DNA will be discussed.

Metal Antitumor Compounds: The Mechanism of Action of Platinum Complexes

Cisplatin (cis-diamminedichloroplatinum(II)) is widely used in the treatment of testicular and ovarian cancers. A number of biological and biochemical results indicate that the reaction of cisplatin with DNA is responsible for the cytotoxic action of this drug. The effect of platinum compounds on the conformation and stability of DNA has been investigated and several platinum-DNA adducts have been identified in vitro and in vivo. Preliminary experiments have quantified the effect of these different lesions on DNA replication, their capacity to induce mutations and their susceptibility to DNA repair processes. Additional DNA damage may be created by platinum(IV) compounds, perhaps during their reduction to platinum(II) compounds by the cell.

Biophysical studies of the modification of DNA by antitumour platinum coordination complexes

Cisplatin (cis-diamminedichloroplatinum(II] is widely used in the treatment of various human tumours. A large body of experimental evidence indicates that the reaction of cisplatin with DNA is responsible for the cytostatic action of this drug. Several platinum-DNA adducts have been identified and their effect on the conformation of DNA has been investigated. Structural studies of platinum-DNA adducts now permit a reasonably good explanation of the biophysical properties of platinated DNA. Antitumouractive platinum compounds induce in DNA, at low levels of binding, local conformational alterations which have the character of non-denaturing distortions. It is likely that these changes occur in DNA due to the formation of intrastrand cross-links between two adjacent purine residues. On the other hand, the modification of DNA by antitumour-inactive complexes results in the formation of more severe local denaturation changes. Conformational alterations induced in DNA by antitumour-active platinum compounds may be reparable with greater difficulty than those induced by the inactive complexes. Alternatively, non-denaturation change induced in DNA by antitumour platinum drugs could represent more significant steric hindrance against DNA replication as compared with inactive complexes.

Palladium(II) compounds with potential antitumour properties and their platinum analogues: a comparative study of the reaction of some orotic acid derivatives with DNA in vitro

Ethidium bromide was used to study perturbations induced in salmon sperm DNA complexed with a series of platinum and palladium compounds obtained from chloro and orotic acid derivatives as leaving ligands. The antitumoral activity of these compounds against Sarcoma 180 cells grafted intraperitoneally into mice is correlated with their capacity to interact with DNA in vitro and to perturb its secondary structure. Nevertheless, among these compounds, [Pt(Dach)(3-methyl-orot)] and [Pt(Dach)(5-fluoro-orot)] do not interact with DNA in vitro and are inactive against Sarcoma 180 cells. This lack of activity originates from the fact that strong chelating properties of the ligand prevent hydrolysis of the compounds which are unable to give rise to aquo species which are the reactive ones. On the other hand, the interaction with DNA is not the only prerequisite in order that a compound be active towards tumour cells. In fact, cis-[Pd(NH3)2Cl2] and cis[Pd(Dach)Cl2] are not antitumoral. It is well known that the former undergoes an inactive trans-conformation and that the two compounds hydrolyse very fast assuming that they interact in vivo with a lot of molecules particularly proteins preventing them to reach the DNA, their pharmacological target. By contrast, [Pd(Dach)(3-methyl-orot)] (T/C = 267%) and [Pd(Dach)(5-fluoro-orot)] (T/C = 270%) display significant antitumour activity.

Relative efficiencies of a series of square-planar platinum(II) compounds on Salmonella mutagenesis

Twelve Pt(II) compounds have been tested for mutagenicity on Salmonella typhimurium (strain TA 100). Very high mutagenic activities were found for the cis derivatives. A correlation is suggested between these results and a formerly described model of chemical reactivity towards DNA, according to which cis derivatives from intra-strand chelates with guanine. A smaller activity was found with monodentate complexes with DNA.

Kinetics of the reaction of cis-platinum compounds with DNA in vitro.

The kinetics of the reaction of a series of cis-platinum(II) compounds with DNA in vitro has been studied using their ability to disturb the secondary structure of the macromolecule. The complexation modifies the stacking of the base pairs and causes an inhibition of the intercalation of ethidium bromide which is correlated with the number of platinum atoms bound per nucleotide. The compounds fall into three groups which react in a few minutes, in a few hours or in several days. The inhibition of the complexation by chloride and carboxylato ions indicates that the interaction occurs through hydrolysed species and that hydrolysis is the rate limiting step. In addition the results indicate that the carboxylato entities are able to react with DNA in vitro without enzymatic activation and that there is no correlation between the antitumoral activity of these compounds against L1210 Leukemia cells and their in vitro reactivity towards DNA.

Replacement of Gln280 by His in TM6 of the human ORL1 receptor increases affinity but reduces intrinsic activity of opioids

The ORL1 ( pioid eceptor‐ ike) receptor is the G protein‐coupled receptor whose amino acid sequence is closest to those of opioid receptors. Residues that are conserved in ORL1 and the three types of opioid receptor, but also a residue, His in the sixth putative transmembrane (TM6) helix, which is present in all opioid receptor types but absent in ORL1, appear to play a key role in receptor recognition and/or activation. Here we have sought to create an opioid binding pocket in the non‐opioid ORL1 receptor by replacing residue Gln280 in its TM6 by the corresponding His residue of opioid receptors. The mutation affects neither the affinity of nociceptin ‐ the natural ORL1 agonist ‐ for the receptor, nor the potency of nociceptin to inhibit adenylyl cyclase via ORL1. In contrast, we find that a few opioid ligands, the agonists lofentanil, etorphine and dynorphin A, and especially the antagonists diprenorphine and nor‐BNI, bind the mutant Q280H receptor with substantially (5‐ to > 100‐fold) higher apparent affinity than they do the wild‐type receptor. Moreover, lofentanil and etorphine no longer act as pure agonists, as they do at the native ORL1 receptor, but are endowed with clear antagonist properties at the mutant receptor. The mutation Q280H, which increases affinity while decreasing intrinsic activity of opioids at ORL1, emphasizes the importance of the His residue for opioid recognition and activation.

Viscosity, nicking, thermal and alkaline denaturation studies on three classes of DNA-platinum complex

Viscosity, nicking, thermal denaturation and alkaline denaturation studies were used to investigate perturbations induced in the DNA secondary structure after complexation with platinum compounds. Three types of DNA-platinum complex, representative of the different modes of platinum binding, have been studied. Cis-Pt(NH3)2Cl2, which forms a cis-bidentate complex with DNA, strongly decreased the viscosity and, according to thermal and alkaline denaturations, destabilized the macromolecule. On the contrary, trans-Pt(NH3)2Cl2, a trans-bidentate complex, stabilized the DNA secondary structure and decreased the viscosity but much less than did cis-Pt(NH3)2Cl2. [Pt(dien)Cl]Cl, a monodentate complex, had no effect on the viscosity; however, addition of this compound stabilized DNA up to 0.01 platinum bound per nucleotide while further Pt binding destabilized the macromolecule. Cis- and trans-Pt(NH3)2Cl2 renatured thermally denatured DNA, which has been interpreted as evidence for the presence of interstrand crosslinks. [Pt(dien)Cl]Cl, on the other hand, did not renature DNA. If the renaturation observed for the bidentate compounds is due only to the presence of interstrand crosslinks, then one interstrand crosslink is found when 400-1000 molecules of the platinum isomer are bound per T7 DNA molecule. Electron microscopy results show that the three types of DNA-platinum complex do not nick DNA up to 0.01 bound platinum per nucleotide.

Correlation between the toxicity of platinum drugs to l1210 leukaemia cells and their mutagenic properties.

Abstract There is a good correlation between the growth inhibitory properties of a series of Pt(II) derivatives on cultured L1210 mouse leukaemia cells and their efficiency of reversion to his + of S. typhimurium TA100, or with their forward mutagenic effects on prophage λ. Reactivity towards DNA may thus explain the antitumor properties of these drugs.