Target-directed discovery and production of pharmaceuticals in transgenic mutant plant cells.

Plants are a source of complex bioactive compounds, with value as pharmaceuticals, or leads for synthetic modification. Many of these secondary metabolites have evolved as defenses against competing organisms and their pharmaceutical value is "accidental", resulting from homology between target proteins in these competitors, and human molecular therapeutic targets. Here we show that it is possible to use mutation and selection of plant cells to re-direct their "evolution" toward metabolites that interact with the therapeutic target proteins themselves. This is achieved by expressing the human target protein in plant cells, and selecting mutants for survival based on the interaction of their metabolome with this target. This report describes the successful evolution of hairy root cultures of a Lobelia species toward increased biosynthesis of metabolites that inhibit the human dopamine transporter protein. Many of the resulting selected mutants are overproducing the active metabolite found in the wild-type plant, but others overproduce active metabolites that are not readily detectable in non-mutants. This technology can access the whole genomic capability of a plant species to biosynthesize metabolites with a specific target. It has potential value as a novel platform for plant drug discovery and production, or as a means of optimizing the therapeutic value of medicinal plant extracts.

Natural Products Genomics: A novel approach for the discovery of anti-cancer therapeutics.

Plants continue to retain some advantages over combinatorial chemistry as sources of novel compounds, for example, they can generate metabolites with a complexity beyond synthetic chemistry. However, this comes with its own problems in production and synthetic modification of these compounds. Natural Products Genomics (NPG) aims to access the plants own genomic capacity to increase yields, and modify complex bioactive metabolites, to alleviate these limitations. NPG uses a combination of gain of function mutagenesis and selection to a) mimic the evolution of novel compounds in plants, and b) to increase yields of known bioactive metabolites. This process is performed rapidly at the cell culture level in large populations of mutants. Two examples demonstrating proof of concept in Nicotiana tabacum (tobacco) and proof of application in the medicinal plant species Catharanthus roseus, are included to illustrate the feasibility of this approach. This biotechnology platform may alter the way in which plant drug discovery is perceived by the pharmaceutical industry, and provides an alternative to combinatorial chemistry for the discovery, modification and production of highly complex bioactive molecules.

Endogenous indoles as novel polyamine site ligands at the N-methyl-D-aspartate receptor complex.

High-throughput ligand displacement screens of a series of endogenous indoles revealed that tryptamine, serotonin and 5-methoxytryptamine readily displace [3H]spermidine and [3H]MK-801 from their respective binding sites in rat brain homogenate. These data, coupled with their potent inhibition of spermidine-potentiated [3H]MK-801 binding, suggest that certain endogenous indoles may act as ligands to one or more polyamine binding sites in the brain, including those on the N-methyl-D-aspartate receptor complex.

Aminoanthraquinones as novel ligands at the polyamine binding site on the N-methyl-D-aspartate receptor complex.

As part of a drug discovery program using high-throughput radioligand-binding assays, aminoanthraquinones were identified as potential modulators of N-methyl-D-aspartate (NMDA) receptor function. Aminoanthraquinones may represent a novel class of polyamine binding site ligands with a unique pharmacophore and may facilitate the rational design of novel NMDA-receptor modulators.

Multiple spinal mediators in parenteral nicotine-induced antinociception.

The role of spinal mechanisms in subcutaneous (s.c.) nicotine-induced antinociception was examined in male Sprague-Dawley rats using the hot-plate and tail-flick tests. Nicotine (0.125, 0.25, 0.375 or 0.5 mg/kg s.c.) produced a dose-related inhibition of nociception in both tests. Although increasing negative geotaxis response times slightly, no significant alteration of other motor reflexes was observed with 0.375 mg/kg of s.c. nicotine. Microinjection of 7 nmol of the high-affinity choline uptake inhibitor hemicholinium-3 into the rostral ventral medulla completely inhibited the antinociception produced by 0.375 mg/kg of s.c. nicotine. Intrathecal (i.t.) injection of 61 nmol of nicotine (in 10 microliter buffer) produced no changes in hot-plate or tail-flick test response latencies. Nicotine-induced antinociception was blocked by a variety of i.t. antagonists injected 12 min before s.c. injection of 0.375 mg/kg of nicotine. In both tests, i.t. pretreatment with 0.1 mumol (in 10 microliter buffer) of scopolamine, methysergide, yohimbine, idazoxan, mecamylamine or 0.2 mumol of atropine attenuated nicotine-induced increases in test response latencies. Pretreatment with 0.1 mumol of atropine attenuated nicotine-induced increases in tail-flick test, but not in the hot-plate test. Pretreatment with 0.1 mumol of i.t. prazosin or naloxone produced no changes in nicotine-induced increases in test response latencies in either test. These data suggest that the antinociception produced by s.c. nicotine is mediated via a number of sites in the spinal cord, including alpha-2 adrenergic, serotonergic and muscarinic cholinergic.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of bergapten and sunlight on cutaneous pigmentation.

The effects of bergapten-containing preparations in sunlight-induced skin pigmentation were evaluated. Oil and lotion vehicles with bergapten/UV-B sunscreen or sunscreen alone were applied to the backs of subjects twice weekly for 4 weeks and the subjects were exposed to gradually increasing doses of midday sunlight. The degree of skin darkening was assessed by clinical examination, reflectometry, and light microscopy of skin biopsy specimens. At 5 weeks, 1 week after the last sunlight exposure, the sites treated with either the bergapten/UV-B sunscreen lotion or the lotion vehicle were significantly darker than the sites treated with the sunscreen lotions without bergapten. Oil preparations produced less clearcut results, possibly because of a less potent sunscreen or because the bergapten did not leave the vehicle and absorb into the epidermis. In type I skin, the bergapten/sunscreen and the oil vehicle alone produced the same amount of tanning; both yielded more tanning than the sunscreen in oil by clinical examination. The findings were not confirmed by reflectometry or by light microscopy. Thus, we conclude that bergapten added to a UV-B sunscreen lotion preparation can increase skin pigmentation over the sunscreen alone when one is exposed to sunlight. The bergapten/UV-B sunscreen combination is a potentially useful product since one can develop a psoralen and UV-A-induced tan while being protected from UV-B-induced sunburn by the UV-B sunscreen incorporated into the formulation.

Characterization of the gene for fructose-1,6-bisphosphatase from Saccharomyces cerevisiae and Schizosaccharomyces pombe. Sequence, protein homology, and expression during growth on glucose.

We have determined the nucleotide sequence of the gene for fructose-1,6-bisphosphatase from both Saccharomyces cerevisiae and Schizosaccharomyces pombe. The predicted protein sequence for fructose-1,6-bisphosphatase from S. cerevisiae contains 347 amino acids and has a molecular weight of 38,100; that from S. pombe, contains 346 amino acids and has a molecular weight of 38,380. Comparison of these amino acid sequences with each other and that of pig kidney fructose-1,6-bisphosphatase shows several regions of strong homology separated by regions of divergence. These homologous regions are likely candidates for functional domains. A gene cassette was constructed for fructose-1,6-bisphosphatase from S. cerevisiae and the gene cassette expressed from the regulated PHO5 and GAL1 promoters of yeast. Yeast cells expressing fructose-1,6-bisphosphatase, while growing on glucose, accumulated large amounts of enzyme intracellularly, suggesting that glucose-regulated proteolytic inactivation does not operate efficiently under these conditions. Growth on glucose was not inhibited by the expression of fructose 1,6-bisphosphatase.

Yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase. Properties of phospho and dephospho forms and of two mutants in which serine 11 has been changed by site-directed mutagenesis.

The properties of dephospho- and phosphofructose-1,6-bisphosphatase from the yeast Saccharomyces cerevisiae and of two mutant enzymes in which the phosphorylatable Ser11 had been changed by site-directed mutagenesis (Ser----Ala and Ser----Asp) were studied to clarify the role of cyclic AMP-dependent phosphorylation of yeast fructose-1,6-bisphosphatase. The mutant enzymes and wild type Ser11 fructose-1,6-bisphosphatase were overexpressed and purified to homogeneity. Phosphofructose-1,6-bisphosphatase was prepared by in vitro phosphorylation. The comparison of the properties of the above enzymes demonstrated that all four had similar maximum activity. However, the phosphoenzyme was about 3-fold more sensitive to AMP and fructose 2,6-bisphosphate inhibition than the dephosphoenzyme, suggesting that regulation operates in vivo by this mechanism, leading to decreased enzyme activity. The purified mutant enzymes Ala11 and Asp11 exhibited properties closely similar to those of dephospho- and phosphofructose-1,6-bisphosphatase, respectively. These results indicate that the functional group at residue 11 is an important factor in the regulation of fructose-1,6-bisphosphatase activity and that Ser(P) can be functionally substituted by Asp in this enzyme.

Reciprocal regulation of the tandemly duplicated PHO5/PHO3 gene cluster within the acid phosphatase multigene family of Saccharomyces cerevisiae.

We characterized the organization and expression of PHO5 and PHO3, the tightly linked repressible and constitutive acid phosphatase genes of Saccharomyces cerevisiae. The "constitutive" gene, PHO3, is expressed only when PHO5 is not. Altering PHO5 expression, either through promoter deletions or through mutations in trans-acting regulatory genes, showed that PHO5 expression is sufficient to block transcription of PHO3. An active genomic copy of PHO5 was able to block expression of PHO3 from a high-copy-number plasmid, showing that some trans-acting product of PHO5 is involved. This is probably a translation product, since the presence of a nontranslatable PHO5 RNA did not inhibit transcription of PHO3.

Antiseptic-induced changes in the cell surface of a chlorhexidine-sensitive and a chlorhexidine-resistant strain of Providencia stuartii.

The effects of chlorhexidine diacetate and benzalkonium chloride on the cell surface of a chlorhexidine-sensitive (Pv 2) and a chlorhexidine-resistant (Pv 67) strain of Providencia stuartii are described. Low concentrations of chlorhexidine diacetate (10 mg/l and upwards) increased the hydrophobicity of Pv 2, whilst having little effect on Pv 67. Both strains were resistant to benzalkonium chloride but a concentration as low as 2 mg/l induced a significant increase in hydrophobicity in Pv 2, with 25-50 mg/l needed to induce a similar type of increase in Pv 67. The possible nature of the resistance is discussed.

Deletions and single base pair changes in the yeast mating type locus that prevent homothallic mating type conversions.

Several cis-acting mutations that prevent homothallic mating type conversions in Saccharomyces cerevisiae have been examined. Deletions within the mating type (MAT) locus were obtained by selecting for survivors among homothallic MAT alpha cells carrying the rad52 mutation. The survivors were unable to switch mating type, even in RAD+ derivatives. The deletions varied in size from fewer than 50 to more than 750 base pairs. All of the deletions removed a Hha I site at the border between the alpha-specific sequences (Y alpha) and the adjacent Z region. We also examined several spontaneous inc mutations that prevent MAT switching. Two of these mutations were cloned in recombinant DNA plasmids and their sequences were determined. The MAT alpha-inc 3-7 mutation proved to have an altered Hha I site at the Y alpha/Z border, by virtue of a single base pair substitution G . C leads to A . T in the second base pair of the Z region (Z2). Restriction fragment analysis showed that two other independently isolated strains with MAT alpha-inc mutations had altered the same Hha I site. The MAT a-inc 4-28 mutation contains a single base pair substitution C . G leads to T . A at position Z6. A base pair difference at position Z11 in two MATa strains does not affect MATa conversions. We conclude that the region near the Y/Z border is essential for the efficient switching of MAT alleles and constitutes an enzyme recognition site for a specific nucleolytic cleavage of MAT DNA.

Acid phosphatase polypeptides in Saccharomyces cerevisiae are encoded by a differentially regulated multigene family.

Two clones from a lambda phage collection containing yeast genes regulated by inorganic phosphate were shown by low-stringency hybridization to select three mRNAs that direct the in vitro synthesis of repressible acid phosphatase (EC 3.1.3.2) polypeptides p60, p58, and p56. By higher stringency hybridization one yeast fragment [8 kilobases (kb)] selects p60 mRNA and the other (5 kb) selects p56 mRNA. These EcoRI digestion fragments were subcloned in yeast transformation vectors and hybridization selection assignments were confirmed by measuring enzyme and mRNA levels in transformants. Enzyme and mRNA levels in (8-kb) high copy number transformants grown in high inorganic phosphate medium revealed a hitherto undetected acid phosphatase protein, P57, which is believed to correspond to the constitutive enzyme encoded by PHO3. The identify of the 8-kb fragment purported to contain the PHO5/PHO3 genes was confirmed by genetic mapping of an integrated copy of this fragment. The site of integration of the 5-kb fragment was demonstrated to be unlinked to the PHO5/PHO3 genes.

Transposition of yeast mating type genes from two translocations of the left arm of chromosome III.

In the yeast Saccharomyces cerevisiae, the HIS4C gene lies on the left arm of chromosome III. We analyzed two chromosomal rearrangements that have HIS4C translocated either to chromosome XII or to a new translocation chromosome. Using the cmt mutation that allows expression of the normally silent copies of mating type genes, we found that both of these translocations also carried HML alpha, more than 30 map units distal to HIS4C which normally lies on chromosome III. In the case of the translocation chromosome (designated T3), we also found an exchange event between HML alpha on the translocation chromosome and HMLa on chromosome III. In diploids containing two T3 chromosomes (one carrying HML alpha and the carrying HMLa), we found that HML was 32 centimorgans from HIS4C, which was 10 centimorgans from an unknown centromere. In homothallic strains carrying HMLa MATa HMRa on chromosome III, switching from MATa to MAT alpha could occur by using the HML alpha on the translocation as the sole donor of alpha information. Transposition from HML alpha on chromosome T3 was about 20 to 40% as efficient as transposition from intact chromosome III. In contrast, transposition from the HML alpha inserted into chromosome XII was reduced about 100-fold. This reduced efficiency did not appear to be caused by an alteration in the sequences immediately surrounding HML alpha in the translocation. The translocated HML alpha sequence was located in the same size (29-kilobase) SalI fragment as was found in chromosome III, and the same EcoRI, HindIII, and BglII restriction sites were also found. Furthermore, HML alpha was still under the control of the CMT gene, which maintains HML as a silent copy of mating type information. These results suggested that the position of the HML alpha sequence plays an important role in the efficiency of mating type switching.

Homothallic conversions of yeast mating-type genes occur by intrachromosomal recombination.

The switching of yeast mating-type alleles involves a transposition of a copy of a sequence from HML or HMR to replace the sequences at MAT. Using diploid strains of yeast we have discovered that about 1% of the homothalic conversions of MAT alleles are accompanied by large intrachromosomal rearrangements. These rearrangements are highly specific fusions of part of MAT either with HMR (to produce a deficiency ring chromosome). We conclude that the mechanism of MAT conversions involves a highly specific pairing between the homologous sequences at MAT and the donor genes HML or HMR followed by a specialized gene conversion event, in which the original allele is replaced by a sequence copied from HMR or HML. At about a 1% frequency conversion of the MAT locus is accompanied by a reciprocal recombination event that results in an intrachromosomal deletion. This same preferential pairing is reflected in a high frequency (> 10(-3)) of site-specific mitotic recombination between MAT alleles on differenat chromosomes. A gene conversion model also allows us to explain the "illegal" transpositions of MAT alleles to HMR or HML that occur when normal excision of MAT is prevented.