Christian M. Apfel

Peptide Deformylase as an Antibacterial Drug Target: Target Validation and Resistance Development

ABSTRACT 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 ofEscherichia 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 theirN-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.

Different agonist- and antagonist-induced conformational changes in retinoic acid receptors analyzed by protease mapping.

The pleiotropic effects of retinoic acid on cell differentiation and proliferation are mediated by two subfamilies of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Recently the synthetic retinoid Ro 41-5253 was identified as a selective RAR alpha antagonist. As demonstrated by gel retardation assays, Ro 41-5253 and two related new RAR alpha antagonists do not influence RAR alpha/RXR alpha heterodimerization and DNA binding. In a limited trypsin digestion assay, complexation of RAR alpha with retinoic acid or several other agonistic retinoids altered the degradation of the receptor such that a 30-kDa proteolytic fragment became resistant to proteolysis. This suggests a ligand-induced conformational change, which may be necessary for the interaction of the DNA-bound RAR alpha/RXR alpha heterodimer with other transcription factors. Our results demonstrate that antagonists compete with agonists for binding to RAR alpha and may induce a different structural alteration, suggested by the tryptic resistance of a shorter 25-kDa protein fragment in the digestion assay. This RAR alpha conformation seems to allow RAR alpha/RXR alpha binding to DNA but not the subsequent transactivation of target genes. Protease mapping with C-terminally truncated receptors revealed that the proposed conformational changes mainly occur in the DE regions of RAR alpha. Complexation of RAR beta, RAR gamma, and RXR alpha, as well as the vitamin D3 receptor, with their natural ligands resulted in a similar resistance of fragments to proteolytic digestion. This could mean that ligand-induced conformational changes are a general feature in the hormonal activation of vitamin D3 and retinoid receptors.

Evaluation of a High-Throughput Fluorescence Assay Method for hERG Potassium Channel Inhibition

The number of projects in drug development that fail in late phases because of cardiac side effects such as QT prolongation can impede drug discovery and development of projects. The molecular target responsible for QT prolongation by a wide range of pharmaceutical agents is the myocardial hERG potassium channel. It is therefore desirable to screen for compound interactions with the hERG channel at an early stage of drug development. Here, the authors report a cell-based fluorescence assay using membrane potential-sensitive fluorescent dyes and stably transfected hERG channels from CHO cells. The assay allows semiautomated screening of compounds for hERG activity on 384-well plates and is sufficiently rapid for testing a large number of compounds. The assay is robust as indicated by a Z′ factor larger than 0.6. The throughput is in the range of 10,000 data points per day, which is significantly higher than any other method presently available for hERG. The data obtained with the fluorescence assay were in qualitative agreement with those from patch-clamp electrophysiological analysis. There were no false-positive hits, and the rate of false-negative compounds is currently 12% but might be further reduced by testing compounds at higher concentration. Quantitative differences between fluorescence and electrophysiological methods may be due to the use- or voltage-dependentactivity of the antagonists.

RARα antagonist RO 41‐5253 inhibits proliferation and induces apoptosis in breast‐cancer cell lines

Ro 41‐5253 is a RARα‐selective antagonist that binds RARα but does not induce transcriptional activation and does not influence RAR/RXR heterodimerization and DNA binding. This retinoid inhibits proliferation and induces apoptosis in MCF‐7 and ZR‐75.1 estrogen‐receptor‐positive breast‐carcinoma cells in a dose‐dependent way. The anti‐proliferative effect is more evident in ZR‐75.1 cells than in MCF‐7 cells and is probably mediated by anti‐AP1 activity, a mechanism known to be implied in the action of several retinoids. In the induction of apoptosis also ZR‐75.1 cells are more sensitive to treatment with Ro 41‐5253 than MCF‐7 cells. In ZR‐75.1 cells an apoptotic/hypodiploid DNA peak is already evident after 2 days of incubation, whereas in MCF‐7 cells it appears only after 4 days. The highest percentage of apoptotic cells, for both cell lines, is reached after 6 days of treatment. The apoptosis pathway is p53‐independent and bcl‐2 down‐regulation seems to be correlated with an increase in TGF‐β1 protein. The MDA‐MB‐231 estrogen‐receptor‐negative cell line is poorly responsive to Ro 41‐5253 treatment, both in terms of proliferation inhibition and apoptosis induction. Ro 41‐5253 has proliferation‐inhibiting and apoptosis‐inducing properties that are not mediated by transcriptional activation from retinoic‐acid response elements. This retinoid antagonist seems to be a compound that exerts an anti‐tumor activity but does not induce the toxic side effects of retinoids and might, therefore, be considered as a candidate for cancer therapy. Int. J. Cancer 78:86–94, 1998.© 1998 Wiley‐Liss, Inc.

Analysis of the ligand-binding domain of human retinoic acid receptor alpha by site-directed mutagenesis.

Three subtypes of retinoic acid receptors (RAR), termed RAR alpha, RAR beta, and RAR gamma, have been described. They are composed of different structural domains, including distinct domains for DNA and ligand binding. RARs specifically bind all-trans-retinoic acid (RA), 9-cis-RA, and retinoid analogs. In this study, we examined the functional role of cysteine and arginine residues in the ligand-binding domain of hRAR alpha (hRAR alpha-LBD, amino acids 154 to 462). All conserved cysteine and arginine residues in this domain were mutated by site-directed mutagenesis, and the mutant proteins were characterized by blocking reactions, ligand-binding experiments, transactivation assays, and protease mapping. Changes of any cysteine residue of the hRAR alpha-LBD had no significant influence on the binding of all-trans RA or 9-cis RA. Interestingly, residue C-235 is specifically important in antagonist binding. With respect to arginine residues, only the two single mutations of R-276 and R-394 to alanine showed a dramatic decrease of agonist and antagonist binding whereas the R272A mutation showed only a slight effect. For all other arginine mutations, no differences in affinity were detectable. The two mutations R217A and R294A caused an increased binding efficiency for antagonists but no change in agonist binding. From these results, we can conclude that electrostatic interactions of retinoids with the RAR alpha-LBD play a significant role in ligand binding. In addition, antagonists show distinctly different requirements for efficient binding, which may contribute to their interference in the ligand-inducible transactivation function of RAR alpha.

Impedance measurement: a new method to detect ligand-biased receptor signaling.

Ligand-biased receptor signaling has been proposed for several G-protein coupled receptors including the niacin receptor GPR109A. Coupling to the G(i/o) pathway has been shown to be responsible for the well described triglyceride lowering effect of nicotinic acid in mice, while activation of the β-arrestin pathway has been suggested to be responsible for its peripheral vasodilatory effect that causes cutaneous flushing. Several ligands have been described to selectively induce triglyceride lowering without inducing flushing. Cellular impedance has been demonstrated to determine G-protein coupled receptors activation in a G-protein specific manner. Agonists, which induce triglyceride lowering, but not flushing show a profile in cellular impedance that is distinct from the one induced by niacin and those compounds that induce triglyceride lowering as well as flushing. The strength of the signal correlates with the activation of β-arrestin.

Direct influence of S9 liver homogenate on fluorescence signals: impact on practical applications in a bacterial genotoxicity assay

Assays based on the bacterial SOS-response offer the possibility of automatization of genotoxicity testing for screening of large compound libraries. While existing assays use colorimetric detection or luminescence read-out, we describe here the use of a fluorescence-based system to achieve high sensitivity of detection required for assay miniaturization. Three commonly used fluorophores--fluorescein, DDAO and resorufin--are evaluated. Experimental evidence is given that S9 liver homogenate contains a heat-labile, reversible fluorophore-binding activity and therefore, significantly reduces fluorescence intensities. We have worked out simple solutions to overcome the S9 related interference in order to be able to establish a robust bacterial genotoxicity assay.

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

ABSTRACT 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.

MUTATIONAL ANALYSIS REVEALS THAT ALL-TRANS-RETINOIC ACID, 9-CIS-RETINOIC ACID, AND ANTAGONIST INTERACT WITH DISTINCT BINDING DETERMINANTS OF RARALPHA

Retinoids exert their pleiotropic effects on cell differentiation and proliferation through specific nuclear receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Two biologically highly active natural retinoids have been identified, all-trans-retinoic acid (t-RA) and 9-cis-retinoic acid (9-cis-RA). The RXRs exclusively bind 9-cis-RA, whereas the RARs bind both isomers of RA with comparable affinity. Recently published results suggest that RARs have the same binding site for t-RA and 9-cis-RA but with different determinants (1–3). Antagonist binding on RARα has been suggested to induce distinct conformational changes in comparison with agonist binding. To elucidate the region minimally required for efficient binding of agonist (t-RA and 9-cis-RA) and antagonist Ro 41-5253 to the RARα, we generated N- and C-terminally truncated mutants of the receptor. Characterization of these deletion mutant proteins using protease mapping and ligand binding experiments revealed that different parts of the ligand-binding domain are necessary for t-RA, 9-cis-RA, and antagonist binding. Three distinct regions of the ligand-binding domain of the human retinoic acid receptor-α are required for binding of t-RA (RARα187–402), 9-cis-RA (RARα188–409), and the antagonist Ro 41-5253 (RARα226–414).