Development of a Photoactivatable Protein Phosphatase-1-Disrupting Peptide.

We describe here the development of a photoreleasable version of a protein phosphatase-1 (PP1)-disrupting peptide (PDP-Nal) that triggers protein phosphatase-1 activity. PDP-Nal is a 23 mer that binds to PP1 through several interactions. It was photocaged on a tyrosine residue, which required the exchange of phenylalanine in PDP-Nal to tyrosine in order to disrupt the most important binding interface. This PDP-caged can be light-controlled in live cells.

Site-selective azide incorporation into endogenous RNase A via a "chemistry" approach.

Site-selective labeling of endogenous proteins represents a major challenge in chemical biology, mainly due to the absence of unique reactive groups that can be addressed selectively. Recently, we have shown that surface-exposed lysine residues of two endogenous proteins and a peptide exhibit subtle changes in their individual reactivities. This feature allows the modification of a single residue in a highly site-selective fashion if kinetically controlled labeling conditions are applied. In order to broaden the scope of the "kinetically-controlled protein labeling" (KPL) approach and highlight additional applications, the water-soluble bioorthogonal reagent, biotin-TEO-azido-NHS (11), is developed which enables the site-selective introduction of an azido group onto endogenous proteins/peptides. This bioconjugation reagent features a biotin tag for affinity purification, an azido group for bioorthogonal labeling, a TEO (tetraethylene oxide) linker acting as a spacer and to impart water solubility and an N-hydroxysuccinimidyl (NHS) ester group for reacting with the exposed lysine residue. As a proof of concept, the native protein ribonuclease A (RNase A) bearing ten available lysine residues at the surface is furnished with a single azido group at Lys 1 in a highly site-selective fashion yielding azido-(K1)RNase A. The K1 site-selectivity is demonstrated by the combined application and interpretation of high resolution MALDI-ToF mass spectroscopy, tandem mass spectroscopy and extracted ion chromatography (XIC). Finally, the water soluble azide-reactive phosphine probe, rho-TEO-phosphine (21) (rho: rhodamine), has been designed and applied to attach a chromophore to azido-(K1)RNase A via Staudinger ligation at physiological pH indicating that the introduced azido group is accessible and could be addressed by other established azide-reactive bioorthogonal reaction schemes.

Definition of the Neurotoxicity-Associated Metabolic Signature Triggered by Berberine and Other Respiratory Chain Inhibitors.

To characterize the hits from a phenotypic neurotoxicity screen, we obtained transcriptomics data for valinomycin, diethylstilbestrol, colchicine, rotenone, 1-methyl-4-phenylpyridinium (MPP), carbaryl and berberine (Ber). For all compounds, the concentration triggering neurite degeneration correlated with the onset of gene expression changes. The mechanistically diverse toxicants caused similar patterns of gene regulation: the responses were dominated by cell de-differentiation and a triggering of canonical stress response pathways driven by ATF4 and NRF2. To obtain more detailed and specific information on the modes-of-action, the effects on energy metabolism (respiration and glycolysis) were measured. Ber, rotenone and MPP inhibited the mitochondrial respiratory chain and they shared complex I as the target. This group of toxicants was further evaluated by metabolomics under experimental conditions that did not deplete ATP. Ber (204 changed metabolites) showed similar effects as MPP and rotenone. The overall metabolic situation was characterized by oxidative stress, an over-abundance of NADH (>1000% increase) and a re-routing of metabolism in order to dispose of the nitrogen resulting from increased amino acid turnover. This unique overall pattern led to the accumulation of metabolites known as biomarkers of neurodegeneration (saccharopine, aminoadipate and branched-chain ketoacids). These findings suggest that neurotoxicity of mitochondrial inhibitors may result from an ensemble of metabolic changes rather than from a simple ATP depletion. The combi-omics approach used here provided richer and more specific MoA data than the more common transcriptomics analysis alone. As Ber, a human drug and food supplement, mimicked closely the mode-of-action of known neurotoxicants, its potential hazard requires further investigation.

Identification of Bioactive Small Molecule Inhibitors of Separase.

Separase, a cysteine protease of the CD clan, triggers chromosome segregation during mitosis by cleaving the cohesin ring entrapping the two sister chromatids. Deregulated separase activity is associated with aneuploidy, a hallmark of most human cancers. In fact, separase is highly overexpressed in many solid cancers, making it an attractive chemotherapeutic target. To identify small molecules capable of inhibiting separase in its complex cellular environment, we established a highly sensitive assay to quantify separase activity in cells and screened a 51 009-member library for separase inhibitors. In vitro assays confirmed that the identified compounds efficiently inhibited separase, while not affecting caspase-1, another CD-clan protease structurally related to separase. Importantly, HeLa cells with compromised separase activity displayed severe chromosome segregation defects upon compound treatment, confirming that the identified inhibitors are bioactive in tumor tissue culture cells. Structure-activity relationship studies succeeded in the optimization of the most promising inhibitor. Overall, this study demonstrates the feasibility of identifying separase-specific inhibitors, which serve as promising lead compounds for the development of clinically relevant separase inhibiting drugs.

Small molecule tools in mitosis research.

Mitosis belongs to the most appealing cellular processes. Yet, the highly dynamic and complex nature of mitosis represents a major challenge when it comes to the functional dissection of mitotic proteins. Due to their fast and often reversible mode of action, small molecules have proven themselves as invaluable tools to dissect mitotic processes. In this chapter, we provide a broad overview of available compounds affecting mitosis. We discuss the different application fields of small molecules and important aspects that have to be considered when using them. Finally, we provide two detailed protocols for the application of small molecules to study mitosis in tissue culture cells.

Theoretical and experimental study of polycyclic aromatic compounds as ß-tubulin inhibitors.

In this work, through a docking analysis of compounds from the ZINC chemical library on human ß-tubulin using high performance computer cluster, we report new polycyclic aromatic compounds that bind with high energy on the colchicine binding site of ß-tubulin, suggesting three new key amino acids. However, molecular dynamic analysis showed low stability in the interaction between ligand and receptor. Results were confirmed experimentally in in vitro and in vivo models that suggest that molecular dynamics simulation is the best option to find new potential ß-tubulin inhibitors. Graphical abstract Bennett's acceptance ratio (BAR) method.