ADN-1184 a monoaminergic ligand with 5-HT(6/7) receptor antagonist activity: pharmacological profile and potential therapeutic utility.

BACKGROUND AND PURPOSE: Many dementia patients exhibit behavioural and psychological symptoms (BPSD) that include psychosis, aggressivity, depression and anxiety. Antipsychotic drugs are frequently prescribed but fail to significantly attenuate mood deficits, may interfere with cognitive function and are associated with motor and cardiac side effects, which are problematic in elderly patients. A need therefore exists for drugs that are better suited for the treatment of BPSD. EXPERIMENTAL APPROACH: We used in vitro cellular and in vivo behavioural tests to characterize ADN-1184, a novel arylsulfonamide ligand with potential utility for treatment of BPSD. KEY RESULTS: ADN-1184 exhibits substantial 5-HT6 /5-HT7 /5-HT2A /D2 receptor affinity and antagonist properties in vitro. In tests of antipsychotic-like activity, it reversed MK-801-induced hyperactivity and stereotypies and inhibited conditioned avoidance response (MED = 3 mg·kg(-1) i.p.). Remarkably, ADN-1184 also reduced immobility time in the forced swim test at low doses (0.3 and 1 mg·kg(-1) i.p.; higher doses were not significantly active). Notably, up to 30 mg·kg(-1) ADN-1184 did not impair memory performance in the passive avoidance test or elicit significant catalepsy and only modestly inhibited spontaneous locomotor activity (MED = 30 mg·kg(-1) i.p.). CONCLUSIONS AND IMPLICATIONS: ADN-1184 combines antipsychotic-like with antidepressant-like properties without interfering with memory function or locomotion. This profile is better than that of commonly used atypical antipsychotics tested under the same conditions and suggests that it is feasible to identify drugs that improve BPSD, without exacerbating cognitive deficit or movement impairment, which are of particular concern in patients with dementia.

Synthesis, anticonvulsant activity and 5-HT1A, 5-HT2A receptor affinity of new N-[(4-arylpiperazin-1-yl)-alkyl] derivatives of 2-azaspiro[4.4]nonane and [4.5]decane-1,3-dione.

The synthesis, physicochemical and pharmacological properties of new N-[(4-arylpiperazin-1-yl)-alkyl]-2-azaspiro[4.4]nonane- (8a-c, 10a-d) and [4.5]decane-1,3-dione (9a-c, 11a-d) derivatives were described. The antiepileptic effects of those compounds were examined by a maximal electroshock (MES) and a pentylenetetrazole (sc. PTZ) tests, and their neurotoxicity was determined using a rota-rod test. Compounds 8c, 9c, 10c, d, 11c, d with a CF(3) group at the 3-position of the 4-arylpiperazine fragment exhibited anti-seizure properties in the MES model; in contrast, their 2-CH(3) and 2-OCH(3) analogues were inactive in both the tests used. Moreover, since the investigated compounds belong to the class of long-chain arylpiperazines, their serotonin 5-HT(1A) and 5-HT(2A) receptor affinity was determined. The relationship between the length of alkylene spacer and 5-HT(1A)/5-HT(2A) receptor activity was observed. Compounds with an ethylene and a propylene bridge (10a-d and 11a-d) were 3-80-fold more potent (K(i) ranged from 3.1 to 94 nM for 5-HT(1A) and 32-465 nM for 5-HT(2A)) than their methylene analogues (8a-c and 9a-c; K(i) ranged from 81 to 370 nM for 5-HT(1A) and 126-1370 nM for 5-HT(2A)). The highest 5-HT(1A) receptor affinity was displayed by 2-OCH(3) and 3-CF(3) phenyl derivatives (10b, 11b: K(i)=6.8 and 5.7 nM, respectively, and 10c, 11c: K(i)=6.0 and 3.1 nM, respectively), while in the case of 5-HT(2A) receptor the highest affinity was observed for the 3-CF(3) phenyl derivatives 10c, d, 11c, d (K(i) ranged from 32 to 86 nM).

Functional dissection of Pdr1p, a regulator of multidrug resistance in Saccharomyces cerevisiae.

Pleiotropic drug resistance in the yeast Saccharomyces cerevisiae results mainly from the overexpression of genes encoding membrane efflux pumps, the so-called ABC and MFS transporters. These pleiotropic drug resistance loci are under the control of the key transcription factors Pdr1p and Pdr3p. We have identified and characterized several new domains of Pdr1p. By testing a series of LexA-PDR1 derivatives for their capacity to activate a GAL1-lacZ reporter gene we have shown that the C-terminal domain of Pdr1p comprising amino acids 879-1036 is involved in transcriptional activation, and that the point mutation pdr1-8 increases its efficiency. Removal of amino acids 1006-1029, which include a polyasparagine stretch, decreases the activation function. Internal deletions within Pdr1p reveal the presence of a large regulatory domain, and a short but strong inhibitory subdomain spanning amino acids 257-316, in which the up-regulating mutations pdr1-2, pdr1-6 and pdr1-7 are located. A mini-Pdr1p consisting of only the DNA-binding and the activation domains strongly up-regulates the expression of the major target genes PDR5, SNQ2 and YOR1, resulting in enhanced multidrug resistance.

The pleitropic drug ABC transporters from Saccharomyces cerevisiae.

The Saccharomyces cerevisiae genome contains 16 genes encoding full-size ABC transporters. Each comprises two nucleotide binding folds (NBF) alternating with transmembrane domains (TM). We have studied in detail three plasma membrane multidrug exporters: Pdr5p (TC3.A.1.205.1) and Snq2p (TC3.A.1.205.2) which share NBF-TM-NBF-TM topology as well as Yor1p (TC3.A.1.208.3) which exhibits the reciprocal TM-NBF-TM-NBF topology. The substrate specificity of Pdr5p, Snq2p and Yor1p are largely, but not totally, overlapping as shown by screening the growth inhibition by 349 toxic compounds of combinatorial deletants of these three ABC genes. Multiple deletion of 7 ABC genes (YOR1, SNQ2, PDR5, YCF1, PDR10, PDR11 and PDR15) and of two transcription activation factors (PDR1 and PDR3) renders the cell from 2 to 200 times more sensitive to numerous toxic coumpounds including antifungals used in agriculture or medicine. The use of the pdr1-3 activating mutation and when necessary of the PDR5 promoter in appropriate multideleted hosts allow high levels of expression of Pdr5p, Snq2p or Yor1 p. These overexpressed proteins exhibit ATPase activity in vitro and confer considerable multiple drug resistance in vivo. The latter property can be used for screening specific inhibitors of fungal and other ABC transporters.

In vivo characterization of the drug resistance profile of the major ABC transporters and other components of the yeast pleiotropic drug resistance network.

Multidrug resistance (MDR) mediated by broad specificity transporters is one of the most important strategies used by pathogens, including cancer cells, to evade chemotherapy. In the yeast Saccharomyces cerevisiae, a complex pleiotropic drug resistance (PDR) network of genes involved in MDR is composed of the transcriptional regulators Pdr1p and Pdr3p, which activate expression of the ATP-binding cassette (ABC) MDR transporters-encoding genes PDR5, SNQ2, and YOR1 as well as other not yet identified genes. We have screened 349 toxic compounds in isogenic S. cerevisiae strains deleted of PDRS, SNQ2, or YOR1 in different combinations as well as both PDR1 and PDR3. The screen revealed extremely promiscuous, yet limited, and to a large extent overlapping but distinct drug resistance profiles of Pdr5p, Snq2p, and Yor1p. These ABC-MDR transporters mediated resistance to most currently available classes of clinically and agriculturally important fungicides and also to many antibiotics, herbicides, and others. Several classes of compounds were identified for the first time in the drug resistance spectrum of MDR transporters. These are fungicides, such as anilinopyrimidines, benzimidazoles, benzenedicarbonitriles, dithiocarbamates, guanidines, imidothiazoles, polyenes, pyrimidynyl carbinols, and strobilurine analogues; the urea derivative and anilide herbicides; flavonoids, several membrane lipids resembling detergents; and newly synthesized lysosomotropic aminoesters; as well as many others. Identification of compounds showing Pdr1p, Pdr3p-dependent, but Pdr5p-, Snq2p-, and Yor1p-independent toxicity, reflected in the case of rhodamine 6G, by efflux alterations, suggests the involvement of new drug resistance genes and is a first step toward their identification. The highly increased toxicity of bile acids toward the PDR1, PDR3 double disruptant together with the decreased level of BAT1 promoter dependent beta-galactosidase activity suggest that the Bat1p ABC transporter is a new member of the PDR network. Our results may contribute to a better understanding of the mechanism of MDR, in particular in the pathogenic yeast Candida albicans. They also provide and indication of the physiological function of MDR transporters and suggest new approaches for the cloning of the mammalian bile acid transporters.

Active efflux by multidrug transporters as one of the strategies to evade chemotherapy and novel practical implications of yeast pleiotropic drug resistance.

Mankind is faced by the increasing emergence of resistant pathogens, including cancer cells. An overview of the different strategies adopted by a variety of cells to evade chemotherapy is presented, with a focus on the mechanisms of multidrug transport. In particular, we analyze the yeast network for pleiotropic drug resistance and assess the potentiality of this system for further understanding of the mechanism of broad specificity and for development of novel practical applications.

Anticancer drugs, ionophoric peptides, and steroids as substrates of the yeast multidrug transporter Pdr5p.

Pdr5p is the yeast Saccharomyces cerevisiae ATP-binding cassette transporter conferring resistance to several unrelated drugs. Its high overproduction in Pdr1p transcription factor mutants allows us to study the molecular mechanism of multidrug transport and substrate specificity. We have developed new in vivo and in vitro assays of Pdr5p-mediated drug transport. We show that in spite of little sequence homology, and inverted topology in respect to that of mammalian P-glycoproteins, Pdr5p shares with them common substrates. Pdr5p extrudes rhodamines 6G and 123, from intact yeast cells in an energy-dependent manner. Plasma membrane preparations from a Pdr5p-overproducing strain exhibit ATP hydrolysis-dependent, osmotically sensitive rhodamine 6G fluorescence quenching. The quenching is competitively inhibited by micromolar concentrations of many anticancer drugs, such as vinblastine, vincristine, taxol, and verapamil, and of ionophoric peptides as well as steroids. In contrast, other anticancer drugs, like colchicine and some multidrug resistance modifiers, such as quinidine, exert noncompetitive inhibition. Our experimental system opens new possibilities for the analysis of structure-function relationship of multidrug transporter substrates and inhibitors.

Solubilization and characterization of the overexpressed PDR5 multidrug resistance nucleotide triphosphatase of yeast.

A 160-kDa plasma membrane protein of the yeast Saccharomyces cerevisiae was overexpressed by mutating the PDR1 or the PDR3 transcription factor gene. The protein is the membrane-bound ATP binding cassette transporter PDR5 (Balzi, E., Wang, M., Leterme, S., Van Dyck, L., and Goffeau, A. (1994) J. Biol. Chem. 269, 2206-2214). PDR5 was solubilized with n-dodecyl-beta-D-malto-side and separated from the PMA1 plasma membrane H(+)-ATPase by glycerol gradient centrifugation. The PDR5 protein hydrolyzes nucleoside diphosphates and triphosphates. This activity is sensitive to low concentrations of vanadate, of oligomycin, and of a variety of hydrophobic compounds. Many of these properties liken PDR5 to the purified mammalian P-glycoprotein responsible for multidrug resistance.