Case study of the automation options and decisions made in implementing a high-throughput cell based screen using the FLIPR.

This case study examines the automation and process change options available to emerging discovery/development stage pharmaceutical companies when considering implementing sophisticated high-throughput screens. Generally there are both financial and personnel constraints that have to be addressed when implementing state-of-the-art screening technology in smaller companies which generally are not as significant as in large pharmaceutical companies. When NPS Pharmaceuticals considered installing a Molecular Devices FLIPR for high-throughput cell based screening it became clear that, to make the best decision, the whole screening process at NPS Pharmaceuticals from screen development and validation, tissue culture, compound distribution, data handling and screening had to be re-examined to see what automation options were possible and which, if any, made sense to implement. Large scale automated systems were not considered due to their cost and the lack of in-house engineering infrastructure to support such systems. The current trend towards workstation based laboratory automation suggested that a minimalist approach to laboratory automation, coupled with improved understanding of the physical process of screening, would yield the best approach. Better understanding of the work flow within the Biomolecular Screening team enabled the group to optimize the process and decide what support equipment was needed. To install the FLIPR, train users, set up the tissue culture protocols for cell supply, establish high-throughput screening database protocols, integrate compound distribution and re-supply and validate the pharmacology on four cell based screens took the team 3 months. The integration of the screening team at the primary, secondary and tertiary screening stages of the target discovery project teams at NPS has enabled us to incorporate minimal automation into the Biomolecular Screening Group whilst retaining an enriching work environment. This is reflected in our current consistent throughput of 64 96-well microplates per day on the FLIPR, a figure that is comparable with that achieved within most major pharmaceutical companies. This case study suggests that process optimization coupled with modern stand alone automated workstations can achieve significant throughput in a resource constrained environment. Significantly greater throughput could be achieved by coupling the process improvement techniques described above with 384-well microplate technology.

5-Hydroxyanthranilic acid derivatives as potent 5-lipoxygenase inhibitors.

Three new 5-lipoxygenase inhibitors, designated as BU-4601 A, B and C, were found in the fermentation broth of Streptomyces sp. strain No. AA2807. Their structures were identified as isodecyl, isoundecyl and isolauryl esters of 5-hydroxyanthranilic acid, respectively. Based on their structures, five related esters were synthesized and evaluated for biological activity as inhibitors of 5-lipoxygenase. Both naturally-occurring and chemically-synthesized compounds exhibited almost equal levels of 5-lipoxygenase inhibitory activities in vitro.

Decreased production of para-hydroxypenicillin V in penicillin V fermentations.

Penicillin V (phenoxymethyl penicillin) is produced by industrial strains of Penicillium chrysogenum in the presence of phenoxyacetic acid (POAc), a side-chain precursor for the penicillin V molecule. The wild-type strain of P. chrysogenum produces an undesirable penicillin byproduct, para-hydroxypenicillin V (p-OH penicillin V), in addition to penicillin V, via para-hydroxylation of POAc and subsequent incorporation of the p-OH phenoxyacetic acid into the penicillin molecule. Most of the p-OH penicillin V is produced late in cycle when the POAc concentration in the medium is nearly depleted. The level of p-OH penicillin V produced by the control strain ranges up to 10-15% of the total penicillins produced. 3-Phenoxypropionic acid and p-bromophenylacetic acid partially inhibit the formation of p-OH penicillin V with a minimal effect on penicillin V productivity. Mutants deficient in their ability to hydroxylate POAc were found to produce lower levels of p-OH penicillin V. Multi-step mutation and screening, starting with the wild-type strain, have culminated in isolation of mutants which produce p-OH penicillin V as 1% of the total penicillins with no adverse effect on penicillin V productivity.

Integrated biological-physicochemical system for the identification of antitumor compounds in fermentation broths.

A rapid, integrated biological-physicochemical system for the identification of six major classes of antitumor compounds in fermentation broths is described. The system relies upon preliminary fractionation of the fermentation broth by liquid-solid extraction, gradient high-performance liquid chromatography with diode-array spectrophotometric detection of the compounds and automated bioassay. The previously stored UV-VIS spectra of the biologically active peaks are used for identification. Confirmation of compound identity is by thermospray liquid chromatography-mass spectrometry. The method has been applied to representatives of the bleomycin, streptonigrin, echinomycin, chromomycin, actinomycin and anthracycline groups.

The biosynthesis of the antibiotic pyrrolnitrin by Pseudomonas aureofaciens.

Feeding experiments with tryptophan samples labeled specifically with radioactive and stable isotopes have shown that Pseudomonas aureofaciens converts this amino acid into pyrrolnitrin in such a way that the indole nitrogen gives rise to the nitro group, the amino group becomes the pyrrole nitrogen, C-3 of the precursor side chain becomes C-3 of the antibiotic, and H-2 of the indole ring and H-alpha of the side chain give rise to H-5 and H-2 of pyrrolnitrin, respectively. Only the L-isomer of tryptophan is incorporated with retention of the alpha-hydrogen and the amino nitrogen. From the D-isomer the labels from these two positions are lost. The obvious conclusion that L-tryptophan is the more immediate precursor is, however, contradicted by the better incorporation of D- than L-tryptophan into the antibiotic. Several potential pathway intermediates were evaluated for incorporation and 4-(0-aminophenyl)-pyrrole was found to be a good precursor. The results area discussed in terms of a plausible pathway for pyrrolnitrin biosynthesis.

Association of 6-oxo-piperidine-2-carboxylic acid with penicillin V. Production on Penicillium chrysogenum fermentations.

Analysis of a 13C NMR spectrum of a concentrated broth from Penicillium chrysogenum fermentation revealed the presence of penicillin V and 6-oxo-piperidine-2-carboxylic acid(1) as the principal constituents. The latter lactam, identical to an authentic sample prepared by the cyclization of alpha-aminoadipic acid was present to the extent of 28 mol% of penicillin V. The lactam isolated form the broth was nearly racemic, having a slight excess of the L-isomer. This isolation provides further evidence regarding the biosynthetic precursors of the hydrophobic penicillins.

Congeners of etamycin produced by Streptomyces griseoviridus.

Streptomyces griseoviridus produces in addition to etamycin several related compounds which can be separated by partition chromatography. One of these has been characterized by amino acid analysis and mass spectrometry and shown to have the same structure as etamycin except for replacement of the hydroxyproline residue by proline. Evidence was obtained for additional congeners similarly related to etamycin by amino acid exchange. The relative proportions of such congeners produced by S. griseoviridus depends upon the medium in which the culture is grown. Certain amino acids support good yields of the metabolites and the culture appears to be steered towards the synthesis of congeners containing such amino acids.

Biosynthesis of the antibiotic 2,5-dihydrophenylalanine by Streptomyces arenae.

The biosynthesis of L-2,5-dihydrophenylalanine (DHPA) in Streptomyces arenae strain Tü 109 was studied in tracer experiments with [U-14C]- and [1,6-14C]shikimic acid followed by chemical degradation of the labeled product. The results indicate that shikimic acid (II) provides only the ring carbons of DHPA, that the side chain of DHPA is attached at the carbon derived from C-1 of II, and that in the transformation of II into DHPA the asymmetry of the ring of II is preserved, with C-6 of II giving rise to C-6' of DHPA. Both generally 14C-labeled chorismate and prephenate, but not L-[3-14C]serine, are incorporated into DHPA. By preparing and feeding 5,6-dihydro[4-3H]prephenate it was shown that this compound is not an intermediate in the biosynthesis of DHPA. A reaction sequence is proposed for the conversion of prephenate to DHPA, involving an allylic rearrangement, followed by 1,4 reduction of the resulting conjugated diene and a combined decarboxylation/dehydration.