Preclinical characterization of WAY-211612: a dual 5-HT uptake inhibitor and 5-HT (1A) receptor antagonist and potential novel antidepressant.

BACKGROUND AND PURPOSE: As a combination of 5-HT selective reuptake inhibitor (SSRI) with 5-HT(1A) receptor antagonism may yield a rapidly acting antidepressant, WAY-211612, a compound with both SSRI and 5-HT(1A) receptor antagonist activities, was evaluated in preclinical models. EXPERIMENTAL APPROACH: Occupancy studies confirmed the mechanism of action of WAY-211612, while its in vivo profile was characterized in microdialysis and behavioural models. KEY RESULTS: WAY-211612 inhibited 5-HT reuptake (K(i) = 1.5 nmol.L(-1); K(B) = 17.7 nmol.L(-1)) and exhibited full 5-HT(1A) receptor antagonist activity (K(i) = 1.2 nmol.L(-1); K(B) = 6.3 nmol.L(-1); I(max) 100% in adenyl cyclase assays; K(B) = 19.8 nmol.L(-1); I(max) 100% in GTPgammaS). WAY-211612 (3 and 30 mg.kg(-1), po) occupied 5-HT reuptake sites in rat prefrontal cortex (56.6% and 73.6% respectively) and hippocampus (52.2% and 78.5%), and 5-HT(1A) receptors in the prefrontal cortex (6.7% and 44.7%), hippocampus (8.3% and 48.6%) and dorsal raphe (15% and 83%). Acute or chronic treatment with WAY-211612 (3-30 mg.kg(-1), po) raised levels of cortical 5-HT approximately twofold, as also observed with a combination of an SSRI (fluoxetine; 30 mg.kg(-1), s.c.) and a 5-HT(1A) antagonist (WAY-100635; 0.3 mg.kg(-1), s.c). WAY-211612 (3.3-30 mg.kg(-1), s.c.) decreased aggressive behaviour in the resident-intruder model, while increasing the number of punished crossings (3-30 mg.kg(-1), i.p. and 10-56 mg.kg(-1), po) in the mouse four-plate model and decreased adjunctive drinking behaviour (56 mg.kg(-1), i.p.) in the rat scheduled-induced polydipsia model. CONCLUSIONS AND IMPLICATIONS: These findings suggest that WAY-211612 may represent a novel antidepressant.

Cloning of a cDNA encoding phosphoenolpyruvate carboxykinase from Haemonchus contortus.

Biochemical and metabolic data have led to the conclusion that the enzyme phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) contributes to a critical point of divergence in energy conservation pathways between mammals and nematodes. To facilitate the determination of the molecular basis for host vs parasite differences in PEPCK, we have cloned a cDNA encoding this enzyme from a parasitic nematode of ruminants, Haemonchus contortus. H. contortus PEPCK was cloned by functional complementation of a PEPCK-, malic enzyme- strain of Escherichia coli (E1786) using an egg stage H. contortus cDNA library in lambda ZAPII. Selection was for growth on malate as the sole carbon source (malate+ phenotype). We isolated a plasmid, pPEPCK, which reproducibly confers a malate+ phenotype in E1786. The sequence of the 2.0-kb EcoRI insert of pPEPCK predicts a 612-amino acid protein which shows about 74% similarity to Drosophila melanogaster and chicken PEPCK. Extracts of E1786[pPEPCK], but not E1786, contain IDP- or GDP-dependent PEPCK enzyme activity. Sequence analysis revealed that the open reading frame (ORF) in pPEPCK lacked a 5' initiation codon and was probably expressed as an in-frame fusion protein with beta-galactosidase. A strategy combining library screening with PCR analysis of positive clones led to the identification of a clone encoding 6 additional NH2-terminal amino acids, including a Met, which, by comparison with known PEPCK amino acid sequences, is likely to be the translation initiation site.

Three beta-tubulin cDNAs from the parasitic nematode Haemonchus contortus.

Experimental evidence indicates that tubulin is the site of action of the anthelmintic benzimidazoles. Furthermore, certain residues of beta-tubulin seem to be critical for this mechanism. Although the benzimidazoles selectively affect nematode vs. mammalian beta-tubulin, the molecular basis for this differential action is not known. To enhance our understanding of this phenomenon, and to provide the basis for investigating benzimidazole resistance in parasitic nematodes, we undertook the cloning of beta-tubulin cDNAs from the ruminant parasite, Haemonchus contortus. We have cloned and sequenced three beta-tubulin cDNAs from this organism, beta 12-16, beta 12-164, and beta 8-9. The first 2 differ at only 23 nucleotides, which give rise to 4 amino acid changes. beta 8-9 represents a different isotype class from the other two, since it differs extensively in the carboxyterminus. By comparing the sequences of these and other nematode beta-tubulins with mammalian beta-tubulins, several regions of consistent difference can be recognized; the functional significance of these regional differences has not been defined. Sequences very similar or identical to beta 8-9 and beta 12-16 are present in both benzimidazole-sensitive and benzimidazole-resistant populations of H. contortus. However, it appears that drug-resistant organisms may differ in the presence of a gene product which is closely related to beta 8-9.

Hair-specific keratins: characterization and expression of a mouse type I keratin gene.

A genomic clone for a member of the mouse type I hair keratin protein family has been isolated and analyzed in order to study the regulation of this keratin during the hair growth cycle. The coding sequence is divided into seven exons. The gene structure is typical of keratins in particular and intermediate filaments in general in that the intron-exon borders are not located at the domain borders of the protein. Comparison with a sheep wool keratin gene shows that the splice sites in the two hair keratin genes are found at identical locations relative to the amino acid sequence of the proteins. Similarly, comparison of the promoter areas of these genes shows several areas of nucleotide sequence conservation, including the area around the TATA box and an SV40 core enhancer sequence. In addition, a high degree of sequence identity exists in the fourth intron. In situ hybridization shows that transcripts of this gene are first found in the relatively undifferentiated proximal cortex area in the keratogenous zone of mouse vibrissae.

Cloning of a cDNA encoding phosphofructokinase from Haemonchus contortus.

Phosphofructokinase (PFK), the key regulatory enzyme in glycolysis, has been cloned from the pathogenic parasitic nematode Haemonchus contortus by functional complementation in Escherichia coli. An E. coli strain deleted for both PFK loci (strain DF1020) was transformed with plasmid DNA from a lambda ZAP II H. contortus cDNA library. Two out of 3 x 10(7) transformants were able to grow on minimal medium with mannitol as the sole carbon source. A plasmid, pPFK, containing a 2.7-kb insert, was isolated from one of these transformants and conferred on DF1020 the ability to grow on mannitol (the PFK phenotype). The complemented cells contain PFK enzyme activity, absent in the E. coli mutant, at levels considerably higher than in wild type E. coli. Sequence analysis of the 2.7-kb insert shows an open reading frame that predicts a 789-amino acid protein that has approximately 70% similarity to mammalian PFKs. The amino acid sequence around asp182, thought to be the catalytic site, is completely conserved from nematodes to mammals.

Single-step purification and biological activity of human nerve growth factor produced from insect cells.

Human nerve growth factor (NGF) was cloned and engineered for expression in a baculovirus-infected Spodoptera frugiperda (SF-9) insect cell system. Culture supernatants contained 2-3 mg/L of recombinant human NGF. The human NGF produced by this system was purified to apparent homogeneity with a single-step affinity chromatography procedure using a high-affinity monoclonal antibody originally raised against murine NGF. The purification procedure yielded 1-2 mg of pure, human NGF per liter of culture supernatant; i.e., approximately 60% recovery of the human NGF originally released into the culture medium. Although the gene transfected into the SF-9 cells coded for pro-NGF, the NGF recovered after purification was greater than 95% fully processed, mature protein. The KD for the affinity of the pure, recombinant human NGF for NGF receptor in PC12 membranes is 0.20 +/- 0.05 nM. Activation of neurite outgrowth in PC12 cells occurs with ED50 values of 85 +/- 20 pM and 9.6 +/- 1.5 pM for a 3-day primary response and a 1-day secondary response, respectively. The pure, recombinant human NGF also stimulates a significant increase in dopamine content of PC12 cells with an ED50 of 5.8 +/- 2.7 pM. These binding and biological activation properties are consistent with values observed using murine NGF purified from submaxillary glands.

DNA sequence recognition by the antitumor antibiotic CC-1065 and analogs of CC-1065.

The DNA base pair preferences of the antitumor antibiotic CC-1065 and two analogs of CC-1065 were studied by following the rate of covalent bond formation (N-3 adenine adduct) with DNA oligomers containing the 5'NNTTA* and 5'NNAAA* sequences (N = nucleotide, A* = alkylated adenine). The rate of adduct formation of CC-1065 is greatly affected by DNA base changes at the fourth and fifth positions of the bonding site for the 5'NNAAA sequences, but not the 5'NNTTA sequences. However, an analog of CC-1065 containing the same alkylating moiety as CC-1065, but not the third fused ring system or additional methylene and oxygen substituents, shows similar rates of adduct formation for all sequences. A second analog of CC-1065 containing three fused ring systems, but not the methylene and oxygen substituents of CC-1065, shows rates of adduct formation with the same sequence dependence as CC-1065, but does not distinguish between the sequences to the degree shown by CC-1065. Adduct formation of CC-1065, but not the analogs, competes with a reversibly bound species. Thymine bases to the 3' side of a potentially reactive adenine or a cytosine base at the fifth position from the bonding adenine create reversible binding sites which decrease the rate of adduct formation of CC-1065. The sequence 5'GCGAATT binds CC-1065 only reversibly. This sequence can compete for CC-1065 with covalent bonding sequences if the sites are located in different oligomers, or if the sites are located (overlapped or not overlapped) in the same oligomer. The results of these competitive binding experiments suggest that the transfer of CC-1065 from the reversible binding site to the covalent bonding site with both sites located on a single DNA duplex, not overlapped, occurs through an equilibrium of CC-1065 in solution, not by migration of CC-1065 in the minor groove.

Escherichia coli expression and processing of human interleukin-1 beta fused to signal peptides.

Escherichia coli expression, processing, and secretion of human interleukin-1 beta (IL-1 beta) fused to the signal peptide of E. coli OmpA or PhoA protein were studied. With fusion to either signal sequence, high-level expression was observed and the products accumulated to about 20% of total cell protein. In the fusion to OmpA leader sequence, more than 50% of the product has the OmpA signal peptide removed precisely. The majority of the processed material is not released by osmotic shock. On the other hand, very little of the material from the fusion to PhoA has the PhoA signal peptide removed. Use of the host with a mutation in prlA or prlF, variation of temperature for cell growth, and alteration of the amino acid residues around the cleavage site do not facilitate processing of the PhoA signal peptide. These results suggest that some component in the PhoA signal peptide, interacting with the Il-1 beta sequence, is interfering with the processing of the signal peptide.

An NMR study of the covalent and noncovalent interactions of CC-1065 and DNA.

The binding of the antitumor drug CC-1065 has been studied with nuclear magnetic resonance (NMR) spectroscopy. This study involves two parts, the elucidation of the covalent binding site of the drug to DNA and a detailed investigation of the noncovalent interactions of CC-1065 with a DNA fragment through analysis of 2D NOE (NOESY) experiments. A CC-1065-DNA adduct was prepared, and an adenine adduct was released upon heating. NMR (1H and 13C) analysis of the adduct shows that the drug binds to N3 of adenine by reaction of its cyclopropyl group. The reaction pathway and product formed were determined by analysis of the 13C DEPT spectra. An octamer duplex, d(CGATTAGC.GCTAATCG), was synthesized and used in the interaction study of CC-1065 and the oligomer. The duplex and the drug-octamer complex were both analyzed by 2D spectroscopy (COSY, NOESY). The relative intensity of the NOEs observed between the drug (CC-1065) and the octamer duplex shows conclusively that the drug is located in the minor groove, covalently attached to N3 of adenine 6 and positioned from the 3'----5' end in relation to strand A [d(CGATTA6GC)]. A mechanism for drug binding and stabilization can be inferred from the NOE data and model-building studies.

Cloning and sequence analysis of the gene encoding invertase from the yeast Schwanniomyces occidentalis.

The gene encoding invertase (INV) has been cloned from Schwanniomyces occidentalis. The enzyme consists of 533 amino acids, 8 potential glycosylation sites and has a 45% identity with the invertase from Saccharomyces cerevisiae. The proenzyme has a 22 amino acid signal sequence that has a high alpha-helical transmembrane potential which differs significantly from that predicted for the Saccharomyces cerevisiae enzyme.

Effect of nucleotide sequences directly downstream from the AUG on the expression of bovine somatotropin in E. coli.

We have studied the expression of bovine somatotropin (BSt) to gain more understanding of various factors affecting translation in E. coli. The unmodified cDNA coding for mature bovine somatotropin does not produce significant amounts of BSt in E. coli using a pBR322-derived vector. However, a translation fusion with 16 codons from trpLE in front of BSt cDNA results in greater than 20% of total cell protein as the fusion product. Analysis of transcription by measuring the rate and integrity of the mRNA confirms that a post-transcriptional event is responsible for the poor expression of the BSt cDNA. There are two potential stem-loop structures in the 5' region of the mRNA which may interfere with translation. To study their effect on translation, lacZ fusions and oligonucleotide mutagenesis were carried out. The results demonstrate that the secondary structure involving the initiation codon blocks translation initiation. Removal of this stem-loop results in a 100-fold increase in BSt expression. However, the expression level is still low, amounting to only 0.5-1% of total cell protein. High level expression can be obtained by replacement of the beginning sequence of BSt cDNA with trpLE codons. These results suggest that in addition to the secondary structure, the nucleotide sequence or amino acid context within the beginning of BSt is incompatible with one of the steps in translation initiation.