Discovery and Characterization of Brigimadlin, a Novel and Highly Potent MDM2-p53 Antagonist Suitable for Intermittent Dose Schedules.

p53 is known as the guardian of the genome and is one of the most important tumor-suppressors. It is inactivated in most tumors, either via tumor protein p53 (TP53) gene mutation or copy number amplification of key negative regulators, e.g., mouse double minute 2 (MDM2). Compounds that bind to the MDM2 protein and disrupt its interaction with p53 restore p53 tumor suppressor activity, thereby promoting cell cycle arrest and apoptosis. Previous clinical experience with MDM2-p53 protein-protein interaction antagonists (MDM2-p53 antagonists) have demonstrated that thrombocytopenia and neutropenia represent on-target dose-limiting toxicities that might restrict their therapeutic utility. Dosing less frequently, while maintaining efficacious exposure, represents an approach to mitigate toxicity and improve the therapeutic window of MDM2-p53 antagonists. However, to achieve this, a molecule possessing excellent potency and ideal pharmacokinetic properties is required. Here, we present the discovery and characterization of brigimadlin (BI 907828), a novel, investigational spiro-oxindole MDM2-p53 antagonist. Brigimadlin exhibited high bioavailability and exposure, as well as dose-linear pharmacokinetics in preclinical models. Brigimadlin treatment restored p53 activity and led to apoptosis induction in preclinical models of TP53 wild-type, MDM2-amplified cancer. Oral administration of brigimadlin in an intermittent dosing schedule induced potent tumor growth inhibition in several TP53 wild-type, MDM2-amplified xenograft models. Exploratory clinical pharmacokinetic studies (NCT03449381) showed high systemic exposure and a long plasma elimination half-life in cancer patients who received oral brigimadlin. These findings support the continued clinical evaluation of brigimadlin in patients with MDM2-amplified cancers, such as dedifferentiated liposarcoma.

High-throughput detection of potential bacteriocin producers in a large strain library using live fluorescent biosensors.

The global increase in antibiotic resistances demands for additional efforts to identify novel antimicrobials such as bacteriocins. These antimicrobial peptides of bacterial origin are already used widely in food preservation and promising alternatives for antibiotics in animal feed and some clinical setting. Identification of novel antimicrobials is facilitated by appropriate high throughput screening (HTS) methods. Previously, we have described a rapid, simple and cost-efficient assay based on live biosensor bacteria for detection of antimicrobial compounds that act on membrane integrity using the ratiometric pH-dependent fluorescent protein pHluorin2 (pHin2). Here, we use these biosensors to develop an integrated pipeline for high-throughput identification of bacteriocin producers and their biosynthetic gene clusters. We extend the existing portfolio of biosensors by generating pHin2 expressing strains of Escherichia coli, Bacillus cereus, Staphylococcus epidermidis, and methicillin-resistant Staphylococcus aureus. These strains were characterized, and control experiments were performed to assess heterogeneity of these biosensors in response to known bacteriocins and develop a robust HTS system. To allow detection of compounds that inhibit target bacteria by inhibiting growth without disturbing membrane integrity, the HTS system was extended with a growth-dependent readout. Using this HTS system, we screened supernatants of a total of 395 strains of a collection of lactic acid bacteria. After two rounds of screening 19 strains of the collection were identified that produced antimicrobial activity against Listeria innocua and Listeria monocytogenes. Genomes of confirmed hits were sequenced and annotated. In silico analysis revealed that the identified strains encode between one and six biosynthetic gene clusters (BGCs) for bacteriocins. Our results suggest that pHin2 biosensors provides a flexible, cheap, fast, robust and easy to handle HTS system for identification of potential bacteriocins and their BGCs in large strain collections.

Two-photon photoemission from CoO layers on Ir(1 0 0).

Two-photon photoelectron spectroscopy is used to study the unoccupied electronic states of cobalt oxide layers on Ir(1 0 0). For thicker layers of (1 0 0) orientation the conduction band minimum is found 2 eV above the Fermi level. Layers with (1 1 1) orientation and thickness ≤4 bilayers show a peak around 3.4 eV energy and no evidence for the conduction band minimum. This is attributed to the metallic character of thin CoO(1 1 1) layers.

Evaluation of the titanium dioxide approach for MS analysis of phosphopeptides.

The affinity of titanium dioxide for phosphate groups has been successfully used for enrichment of phosphopeptides from complex mixtures. This paper reports the relationship between the occurrence of some amino acids and the phospho-specific and nonspecific binding of peptides that occurs during titanium dioxide enrichment. In order to perform a systematic study, two well-characterized peptide mixtures consisting of either 33 or 8 synthetic phosphopeptides and their nonphosphorylated analogs, which differed in charge and hydrophobicity, were synthesized and analyzed by ESI-MS and MALDI-MS. The titanium dioxide procedure was also evaluated for comprehensive detection of phosphopeptides in phosphoproteomics. In summary, our results clearly confirm the high selectivity of titanium dioxide for phosphorylated sequences. Drastically reduced recovery was observed for phosphopeptides with multiple basic amino acids. Nonspecific binding of nonphosphorylated peptides and sample loss of phosphopeptides must also be taken into account.