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Autophagy mediates the degradation of damaged proteins, organelles and pathogens, and plays a key role in health and disease. Thus, the identification of new mechanisms involved in the regulation of autophagy is of major interest. In particular, little is known about the role of lipids and lipid-binding proteins in the early steps of autophagosome biogenesis. Using target-agnostic, high-content, image-based identification of indicative phenotypic changes induced by small molecules, we have identified autogramins as a new class of autophagy inhibitor. Autogramins selectively target the recently discovered cholesterol transfer protein GRAM domain-containing protein 1A (GRAMD1A, which had not previously been implicated in autophagy), and directly compete with cholesterol binding to the GRAMD1A StART domain. GRAMD1A accumulates at sites of autophagosome initiation, affects cholesterol distribution in response to starvation and is required for autophagosome biogenesis. These findings identify a new biological function of GRAMD1A and a new role for cholesterol in autophagy.

Aminoferrocene-based prodrugs are activated in cancer cells by reactive oxygen species (ROS). They were shown to exhibit high cytotoxicity towards a variety of cancer cell lines and primary cancer cells, but remain not toxic towards non-malignant cells. However, these prodrugs have rather high lipophilicity leading to relatively low water solubility. In particular, an n-octanol/water partition coefficient for the best aminoferrocene-based prodrug (2) was found to be 4.51±0.03. Though the approaches for decreasing lipophilicity are straightforward and include the addition of polar residues to the drug structure, these modifications also lead to dramatic decrease of cell permeability and, correspondingly, lower the activity of the drug. Therefore, a delicate balance of polar and unpolar groups should be found to reduce lipophilicity without compromising the useful drug properties. In this study we optimized an N-alkyl substituent, which is a key element responsible for the stabilization of the aminoferrocene drug released in cancer cells from prodrug 2. We found that an N-propargyl residue is an optimal replacement for the N-benzyl fragment. In particular, such a substitution (prodrug 7a) leads to reduction of prodrug lipophilicity down to logP=3.78±0.05, improvement of its water solubility, decrease of its propensity towards aggregation and dramatic increase of its ROS-generating properties. Finally, we demonstrated that the optimized prodrug strongly suppresses growth of Guerin's carcinoma (T8) in vivo at the dose of 30mg/kg.

N-Terminal myristoylation facilitates membrane binding and activity of proteins, in particular of Src family kinases, but the underlying mechanisms are only beginning to be understood. The chaperones UNC119A/B regulate the cellular distribution and signaling of N-myristoylated proteins. Selective small-molecule modulators of the UNC119-cargo interaction would be invaluable tools, but have not been reported yet. We herein report the development of the first UNC119-cargo interaction inhibitor, squarunkin A. Squarunkin A selectively inhibits the binding of a myristoylated peptide representing the N-terminus of Src kinase to UNC119A with an IC50 value of 10 nm. It binds to UNC119 proteins in cell lysate and interferes with the activation of Src kinase. Our results demonstrate that small-molecule inhibition of the UNC119-cargo interaction might provide new opportunities for modulating the activity of Src kinases that are independent of direct inhibition of the enzymatic kinase activity.

The acyl‐binding UNC119 proteins mediate the activation and transport of various N‐myristoylated proteins. In particular, UNC119a plays a crucial role in the completion of cytokinesis. Herein, we report the use of a lipidated peptide originating from the UNC119 binding partner Gnat1 as the basis for the design of lipidated, stabilized α‐helical peptides that target UNC119a. By using the hydrocarbon peptide‐stapling approach, cell‐permeable binders of UNC119a were generated that induced the accumulation of cytokinetic and binucleated cells; this suggests UNC119a as a potential target for the inhibition of cytokinesis., Targeting molecular chaperones: Cell‐permeable peptidomimetics of a myristoylated peptide were obtained by hydrocarbon peptide stapling. These agents target the human acyl‐binding protein UNC119a, which plays a crucial role in cytokinesis, inducing the accumulation of cytokinetic and binucleated cells. This suggests UNC119a as a potential target for the inhibition of cytokinesis.

Aminoferrocene-based prodrugs are activated in the presence of cancer-specific amounts of reactive oxygen species, e.g. H2O2, with the formation of products of two types: Fe-containing complexes, which catalyze generation of HO and O2(-), and quinone methides, which alkylate glutathione and inhibit the antioxidative system of the cell. Both processes act synergistically by increasing the oxidative stress in cancer cells thereby leading to their death. However, in the activation step including the cleavage of a B-C bond one molecule of H2O2 is consumed that counteracts the desired effect of the products released from aminoferrocenes. We replaced an H2O2-sensitive trigger in original prodrugs with an azide group. This trigger is slowly reduced in the presence of glutathione with the formation of an unstable arylamine intermediate, which decomposes with the release of iron ions and iminoquinone methides. These products induce strong oxidative stress in cells as we confirmed using 2',7'-dichlorodihydrofluorescin diacetate reagent in combination with flow cytometry. In this case the activation process does not consume H2O2. Correspondingly, we observed that the azide-containing prodrug is substantially more toxic towards human promyelocytic leukemia cell line HL-60 (IC50=27±4μM) than its H2O2-responsive analogue (IC50>50μM).

An innovative production technique is described for the low-cost production of arrays of interdigitated electrode (IDE) structures. The resulting polymer chips form the basis of a new type of diagnostic device allowing for the impedimetric detection of either hybridisation or immuno-affinity binding of antibody-antigen combinations. Sensitivity of the IDEs is maximised by focusing the field of sensitivity to the molecules of interest by lowering the electrode dimensions and spacing to the sub-micron region. This has been achieved using a unique combination of state-of-the-art micro-structuring of polymers and the directionality of metal-deposition by evaporation. Subsequently, polymer based arrays of IDEs with micron to sub-micron electrode widths can be realised using a single metallisation step, completely omitting any sophisticated photolithography.

We describe a simple and sensitive method for enhancement of the silver-gold-intensified 3,3'-diaminobenzidine (DAB) reaction demonstrating peroxidase activity. After completing silver-gold intensification of the preparations immunostained by the avidin-biotin-peroxidase method with DAB as the chromogen, the preparations were immersed in a solution containing uranyl nitrate. This new method appeared to increase the sensitivity by at least one order of magnitude as compared with silver-gold intensification alone.