Phase I study of RGB-286638, a novel, multitargeted cyclin-dependent kinase inhibitor in patients with solid tumors.

PURPOSE: RGB-286638 is a multitargeted inhibitor with targets comprising the family of cyclin-dependent kinases (CDK) and a range of other cancer-relevant tyrosine and serine/threonine kinases. The objectives of this first in human trial of RGB-286638, given i.v. on days 1 to 5 every 28 days, were to determine the maximum tolerated dose (MTD) and to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of this new drug. EXPERIMENTAL DESIGN: Sequential cohorts of 3 to 6 patients were treated per dose level. Blood, urine samples, and skin biopsies for full PK and/or PD analyses were collected. RESULTS: Twenty-six patients were enrolled in 6-dose levels from 10 to 160 mg/d. Four dose-limiting toxicities were observed in 2 of the 6 patients enrolled at the highest dose level. These toxicities were AST/ALT elevations in 1 patient, paroxysmal supraventricular tachycardias (SVTs), hypotension, and an increase in troponin T in another patient. The plasma PK of RGB-286638 was shown to be linear over the studied doses. The interpatient variability in clearance was moderate (variation coefficient 7%-36%). The PD analyses in peripheral blood mononuclear cells, serum (apoptosis induction) and skin biopsies (Rb, p-Rb, Ki-67, and p27(KIP1) expression) did not demonstrate a consistent modulation of mechanism-related biomarkers with the exception of lowered Ki-67 levels at the MTD level. The recommended MTD for phase II studies is 120 mg/d. CONCLUSIONS: RGB-286638 is tolerated when administered at 120 mg/d for 5 days every 28 days. Prolonged disease stabilization (range, 2-14 months) was seen across different dose levels.

Prediction of mechanisms of action of antibacterial compounds by gene expression profiling.

We have generated a database of expression profiles carrying the transcriptional responses of the model organism Bacillus subtilis following treatment with 37 well-characterized antibacterial compounds of different classes. The database was used to build a predictor for the assignment of the mechanisms of action (MoAs) of antibacterial compounds by the use of support vector machines. This predictor was able to correctly classify the MoA class for most compounds tested. Furthermore, we provide evidence that the in vivo MoA of hexachlorophene does not match the MoA predicted from in vitro data, a situation frequently faced in drug discovery. A database of this kind may facilitate the prioritization of novel antibacterial entities in drug discovery programs. Potential applications and limitations are discussed.

Panel of Bacillus subtilis reporter strains indicative of various modes of action.

In a recent project, we collected the transcriptional profiles of Bacillus subtilis 168 after treatment with a large set of diverse antibacterial agents. One result of the data analysis was the identification of marker genes that are indicative of certain compounds or compound classes. We cloned these promoter regions in front of a luciferase reporter gene and reintroduced the constructs individually into the B. subtilis chromosome. Strains were analyzed for their responsiveness after treatment with a set of 37 antibacterials. Twelve functional reporter strains were generated that were selectively and significantly upregulated by the compounds. The selectivity of the reporter strains ranged from generic pathways like protein biosynthesis, cell wall biosynthesis, and fatty acid biosynthesis to compound classes (quinolones and glycopeptides) and individual compounds (rifampin, cycloserine, and clindamycin). Five of the strains are amenable for high-throughput applications, e.g., pathway-specific screening. In summary, we successfully generated B. subtilis reporter strains that are indicative of the mechanisms of action of various classes of antibacterials. The set of reporter strains presented herein can be used for mode-of-action analyses and for whole-cell screening of compound libraries in a mode-of-action-specific manner.

Identification by RNA profiling and mutational analysis of the novel copper resistance determinants CrdA (HP1326), CrdB (HP1327), and CzcB (HP1328) in Helicobacter pylori.

Mechanisms involved in maintaining cytoplasmic metal ion homeostasis play a central role in the adaptation of Helicobacter pylori to the changing gastric environment. An investigation of the global regulatory responses to copper ions by using RNA profiling with a threshold factor of 4.0 revealed that copper induces transcription of 19 H. pylori genes and that only the ferritin gene pfr is repressed. The 57-fold copper induction identified the HP1326 gene encoding an H. pylori-specific protein as a candidate for a novel copper resistance determinant. The HP1326 gene is expressed as a monocistronic unit, and two small HP1326 mRNAs are copper induced. The HP1326 protein is secreted and is required for copper resistance maintained by cytoplasmic copper homeostasis, as H. pylori HP1326 mutants were copper sensitive and displayed increased copper induction of HP1326 transcription as well as elevated copper repression of ferritin synthesis. The clear copper-sensitive phenotype displayed by H. pylori HP1327 and HP1328 mutants provides strong evidence that the HP1326 protein, together with the signal peptide site of the H. pylori-specific protein HP1327, whose gene is located downstream from that encoding HP1326, and the CzcB and CzcA metal efflux system component homologs HP1328 and HP1329, constitutes a novel type of copper efflux pump, as discussed below. The HP1329 gene could not be inactivated, but the 14-fold transcriptional copper induction determined by RNA profiling points towards a function of the encoded CzcA homolog in copper resistance. In summary, results from RNA profiling identified the novel H. pylori-specific copper resistance determinants CrdA (HP1326) and CrdB (HP1327), which are required for adaptation to copper-rich environmental conditions.