Extensive targeting of chemical space at the prime side of ketoamide inhibitors of rhomboid proteases by branched substituents empowers their selectivity and potency.

Rhomboid intramembrane serine proteases have been implicated in several pathologies, and emerge as attractive pharmacological target candidates. The most potent and selective rhomboid inhibitors available to date are peptidyl α-ketoamides, but their selectivity for diverse rhomboid proteases and strategies to modulate it in relevant contexts are poorly understood. This gap, together with the lack of suitable in vitro models, hinders ketoamide development for relevant eukaryotic rhomboid enzymes. Here we explore the structure-activity relationship principles of rhomboid inhibiting ketoamides by medicinal chemistry and enzymatic in vitro and in-cell assays with recombinant rhomboid proteases GlpG, human mitochondrial rhomboid PARL and human RHBDL2. We use X-ray crystallography in lipidic cubic phase to understand the binding mode of one of the best ketoamide inhibitors synthesized here containing a branched terminal substituent bound to GlpG. In addition, to extend the interpretation of the co-crystal structure, we use quantum mechanical calculations and quantify the relative importance of interactions along the inhibitor molecule. These combined experimental analyses implicates that more extensive exploration of chemical space at the prime side is unexpectedly powerful for the selectivity of rhomboid inhibiting ketoamides. Together with variations in the peptide sequence at the non-prime side, or its non-peptidic alternatives, this strategy enables targeted tailoring of potent and selective ketoamides towards diverse rhomboid proteases including disease-relevant ones such as PARL and RHBDL2.

Chemical Blockage of the Mitochondrial Rhomboid Protease PARL by Novel Ketoamide Inhibitors Reveals Its Role in PINK1/Parkin-Dependent Mitophagy.

The mitochondrial rhomboid protease PARL regulates mitophagy by balancing intramembrane proteolysis of PINK1 and PGAM5. It has been implicated in the pathogenesis of Parkinson's disease, but its investigation as a possible therapeutic target is challenging in this context because genetic deficiency of PARL may result in compensatory mechanisms. To address this problem, we undertook a hitherto unavailable chemical biology strategy. We developed potent PARL-targeting ketoamide inhibitors and investigated the effects of acute PARL suppression on the processing status of PINK1 intermediates and on Parkin activation. This approach revealed that PARL inhibition leads to a robust activation of the PINK1/Parkin pathway without major secondary effects on mitochondrial properties, which demonstrates that the pharmacological blockage of PARL to boost PINK1/Parkin-dependent mitophagy is a feasible approach to examine novel therapeutic strategies for Parkinson's disease. More generally, this study showcases the power of ketoamide inhibitors for cell biological studies of rhomboid proteases.

Light-Activatable, 2,5-Disubstituted Tetrazoles for the Proteome-wide Profiling of Aspartates and Glutamates in Living Bacteria.

Covalent inhibitors have recently seen a resurgence of interest in drug development. Nevertheless, compounds, which do not rely on an enzymatic activity, have almost exclusively been developed to target cysteines. Expanding the scope to other amino acids would be largely facilitated by the ability to globally monitor their engagement by covalent inhibitors. Here, we present the use of light-activatable 2,5-disubstituted tetrazoles that allow quantifying 8971 aspartates and glutamates in the bacterial proteome with excellent selectivity. Using these probes, we competitively map the binding sites of two isoxazolium salts and introduce hydrazonyl chlorides as a new class of carboxylic-acid-directed covalent protein ligands. As the probes are unreactive prior to activation, they allow global profiling even in living Gram-positive and Gram-negative bacteria. Taken together, this method to monitor aspartates and glutamates proteome-wide will lay the foundation to efficiently develop covalent inhibitors targeting these amino acids.

A Pronucleotide Probe for Live-Cell Imaging of Protein AMPylation.

Conjugation of proteins to AMP (AMPylation) is a prevalent post-translational modification (PTM) in human cells, involved in the regulation of unfolded protein response and neural development. Here we present a tailored pronucleotide probe suitable for in situ imaging and chemical proteomics profiling of AMPylated proteins. Using straightforward strain-promoted azide-alkyne click chemistry, the probe provides stable fluorescence labelling in living cells.

Physical therapy is targeted and adjusted over time for the rehabilitation of locomotor function in acute spinal cord injury interventions in physical and sports therapy.

STUDY DESIGN: Prospective observational multicenter study. OBJECTIVES: Investigation of content, duration and adjustment of physical therapy for the rehabilitation of ambulation in acute spinal cord injury (SCI). SETTING: European Multicenter Study of SCI (EMSCI). METHODS: Physical therapy interventions during acute in-patient rehabilitation of eighty incomplete SCI patients (AIS B, C, D all lesion levels) were recorded using the SCI - Intervention Classification System. Mobility was documented using the Spinal Cord Independence Measurement (SCIM III), demographics and clinical data were retrieved from the EMSCI database. RESULTS: Overall recovery of locomotor function was categorized into three outcome groups (G1-G3). Of 76 initial wheelchair-using patients, 53.9% remained wheelchair user (G1), 25% regained moderate (G2) and 21.1% good walking (G3) capability. Strength training was the most frequently applied intervention of body function/-structure across all outcome groups (about 30% of all interventions), while interventions focusing on muscle tone and respiration were predominantly applied in wheelchair-dependent patients. Activity-focused interventions of transfer, transition, sitting were trained most intensively in outcome group G1, while walking and swimming were increasingly trained in patients with moderate and good walking outcomes. Physical therapy interventions of assistive and active trainings as well as corresponding training environments changed with the recovery of locomotor function. CONCLUSIONS: Physical therapy of locomotor function is targeted to individual patients' conditions and becomes adjusted to the progress of ambulation. Although the involved clinical sites were not following explicitly standardized rehabilitation programs, common patterns can be discerned which may form the basis of prospective standardized programs.

The effects of TORC signal interference on lipogenesis in the oleaginous yeast Trichosporon oleaginosus.

BACKGROUND: Oleaginous organisms are a promising, renewable source of single cell oil. Lipid accumulation is mainly induced by limitation of nutrients such as nitrogen, phosphorus or sulfur. The oleaginous yeast Trichosporon oleaginosus accumulates up to 70% w/w lipid under nitrogen stress, while cultivation in non-limiting media only yields 9% w/w lipid. Uncoupling growth from lipid accumulation is key for the industrial process applicability of oleaginous yeasts. This study evaluates the effects of rapamycin on TOR specific signaling pathways associated with lipogenesis in Trichosporon oleaginosus for the first time. RESULTS: Supplementation of rapamycin to nutrient rich cultivation medium led to an increase in lipid yield of up to 38% g/L. This effect plateaued at 40 µM rapamycin. Interestingly, the fatty acid spectrum resembled that observed with cultivation under nitrogen limitation. Significant changes in growth characteristics included a 19% increase in maximum cell density and a 12% higher maximum growth rate. T. oleaginosus only has one Tor gene much like the oleaginous yeast Rhodosporidium toruloides. Consequently, we analyzed the effect of rapamycin on T. oleaginosus specific TORC signaling using bioinformatic methodologies. CONCLUSIONS: We confirm, that target of rapamycin complex 1 (TORC1) is involved in control of lipid production and cell proliferation in T. oleaginosus and present a homology based signaling network. Signaling of lipid induction by TORC1 and response to carbon depletion to this complex appear to be conserved, whereas response to nitrogen limitation and autophagy are not. This work serves as a basis for further investigation regarding the control and induction of lipid accumulation in oil yeasts.