2-(2-Oxo-1,4-dihydro-2H-quinazolin-3-yl)- and 2-(2,2-dioxo-1,4-dihydro-2H-2lambda6-benzo[1,2,6]thiadiazin-3-yl)-N-hydroxy-acetamides as potent and selective peptide deformylase inhibitors.

Potent, selective, and structurally new inhibitors of the Fe(II) enzyme Escherichia coli peptide deformylase (PDF) were obtained by rational optimization of the weakly binding screening hit (5-chloro-2-oxo-1,4-dihydro-2H-quinazolin-3-yl)-acetic acid hydrazide (1). Three-dimensional structural information, gathered from Ni-PDF complexed with 1, suggested the preparation of two series of related hydroxamic acid analogues, 2-(2-oxo-1,4-dihydro-2H-quinazolin-3-yl)-N-hydroxy-acetamides (A) and 2-(2,2-dioxo-1,4-dihydro-2H-2lambda(6)-benzo[1,2,6]thiadiazin-3-yl)-N-hydroxy-acetamides (B), among which potent PDF inhibitors (37, 42, and 48) were identified. Moreover, two selected compounds, one from each series, 36 and 41, showed good selectivity for PDF over several endoproteases including matrix metalloproteases. However, these compounds showed only weak antibacterial activity.

Hydroxamic acid derivatives as potent peptide deformylase inhibitors and antibacterial agents.

Low-molecular-weight beta-sulfonyl- and beta-sulfinylhydroxamic acid derivatives have been synthesized and found to be potent inhibitors of Escherichia coli peptide deformylase (PDF). Most of the compounds synthesized and tested displayed antibacterial activities that cover several pathogens found in respiratory tract infections, including Chlamydia pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. The potential of these compounds as antibacterial agents is discussed with respect to selectivity, intracellular concentrations in bacteria, and potential for resistance development.

Phenylalanine hydroxylase from human kidney.

In this report the presence, and level, of phenylalanine hydroxylase in the cortex of human kidney is established. The average activity found in 15 surgically removed kidneys was 47.2 plus or minus 11.2 mU/g wet weight of tissue. The average value, determined under the same experimental conditions, for two human liver biopsies was 217 mU/g tissue. Of five autopsy livers obtained 2.5-4 h postmortem, four contained no activity, and only 1-2 percent of normal was found in the fifth. Autopsy kidneys were similarly inactive. The presence of a highly active degradative enzyme could not be demonstrated in autopsy liver homogenates; it was established that the lack of activity was not due to an inhibitory component. A possible interpretation of this phenomenon is discussed. According to work published elsewhere [13] the kidney and liver enzymes appear to be similar. Thus, surgically removed kidneys provide an alternative source of human phenylalanine hydroxylase which can be used to study phenylketonuria.

Peptide deformylase as an antibacterial drug target: assays for detection of its inhibition in Escherichia coli cell homogenates and intact cells.

An assay was developed to determine the activity of peptide deformylase (PDF) inhibitors under conditions as close as possible to the physiological situation. The assay principle is the detection of N-terminal [35S]methionine labeling of a protein that contains no internal methionine. If PDF is active, the deformylation of the methionine renders the peptide a substrate for methionine aminopeptidase, resulting in the removal of the N-terminal methionine label. In the presence of a PDF inhibitor, the deformylation is blocked so that the N-formylated peptide is not processed and the label is detected. Using this assay, it is possible to determine the PDF activity under near-physiological conditions in a cell-free transcription-translation system as well as in intact bacterial cells.

The effect of a new specific alpha-amylase inhibitor on post-prandial glucose and insulin excursions in normal subjects and Type 2 (non-insulin-dependent) diabetic patients.

Trestatin (Ro 9-0154), a new specific alpha-amylase inhibitor of microbial origin, was tested in six normal subjects and seven Type 2 (non-insulin-dependent) diabetic patients. In normal subjects the maximal increases in blood glucose following a 115-g starch meal were 2.19 +/- 0.57 mmol/l (mean +/- SEM) with placebo, but 1.32 +/- 0.39 mmol/l with 10 mg, 1.06 +/- 0.26 mmol/l with 20 mg, 0.43 +/- 0.07 mmol/l with 50 mg (p less than 0.05) and 0.26 +/- 0.14 mmol/l with 100 mg (p less than 0.05) Trestatin . The corresponding increases in plasma insulin were 116.5 +/- 19.6 mU/l; 74.8 +/- 17.5 mU/l; 50.7 +/- 8.3 mU/l; 28.7 +/- 6.9 mU/l (p less than 0.05) and 16.5 +/- 3.2 mU/l (p less than 0.05). In the diabetic patients the maximal increases in blood glucose following a 50-g starch meal were 6.09 +/- 0.02 mmol/l with placebo, but 3.17 +/- 0.59 mmol/l (p less than 0.05) with 10 mg and 1.69 +/- 0.41 mmol/l (p less than 0.05) with 30 mg Trestatin . The corresponding insulin increases were: 58.8 +/- 12.7 mU/l, 31.5 +/- 9.7 mU/l (p less than 0.05) and 23.4 +/- 4.8 mU/l (p less than 0.05). Trestatin fully retained this pharmacological activity during treatment for 4 weeks in the diabetic patients. Trestatin did not influence glucose and insulin profiles after oral glucose and sucrose. These results are consistent with a specific inhibition of alpha-amylase in man.

A retinoic acid receptor alpha antagonist selectively counteracts retinoic acid effects.

Retinoic acid (RA) exerts its pleiotropic effects on cell growth and differentiation through the activation of a family of transcription factors-the RA receptors (RARs). Three subtypes of these receptors exist, RAR alpha, RAR beta, and RAR gamma. The receptors are differentially expressed in different cell types and stages of development, suggesting that they may regulate different sets of genes. We have identified a synthetic retinoid with the characteristics of a selective RAR alpha antagonist. This antagonist counteracts RA effects on HL-60 cell differentiation and on B-lymphocyte polyclonal activation. Beyond its potential practical relevance, this and other specific antagonists will be useful to dissect the RAR system and to assign to one given receptor each of the many RA-regulated functions.

Peptide deformylase as an antibacterial drug target: target validation and resistance development.

New inhibitors of peptide deformylase (PDF) which are very potent against the isolated enzyme and show a certain degree of antibacterial activity have recently been synthesized by our group. Several lines of experimental evidence indicate that these inhibitors indeed interfere with the target enzyme in the bacterial cell. (i) The inhibition of Escherichia coli growth could be counteracted by overexpression of PDF from different organisms, including E. coli, Streptococcus pneumoniae, and Haemophilus influenzae. Conversely, reduced expression of PDF in S. pneumoniae resulted in an increased susceptibility to the inhibitors. (ii) Proteome analysis on two-dimensional gels revealed a shift for many proteins towards lower pI in the presence of PDF inhibitors, as would be expected if the proteins still carry their N-formyl-Met terminus. (iii) PDF inhibitors show no antimicrobial activity against E. coli under conditions that make growth independent of formylation and deformylation. The antibacterial activity in E. coli was characterized as bacteriostatic. Furthermore, the development of resistance in E. coli was observed to occur with high frequency (10(-7)). Resistant mutants show a reduced growth rate, and DNA sequence analysis revealed mutations in their formyl transferase gene. Taking all these aspects into account, we conclude that PDF may not be an optimal target for broad-spectrum antibacterial agents.