Isonitrile-Proline - A Versatile Handle for the Chemoselective Derivatization of Collagen Peptides.

Functional groups that allow for chemoselective and bioorthogonal derivatization are valuable tools for the labelling of peptides and proteins. The isonitrile is such a group but synthetic methods for its incorporation into peptides by solid-phase peptide synthesis are not known. Here, we introduce (4S)- and (4R)-isonitrileproline (Inp) as building blocks for solid-phase peptide synthesis. Conformational studies of (4S)- and (4R)-Inp and thermal stability analysis of Inp-containing collagen triple helices revealed that the isonitrile group exerts a stereoelectronic gauche effect. We showcase the value of Inp for bioorthogonal labelling by derivatization of Inp-containing collagen model peptides (CMPs). Dual labelling with a pair of bioorthogonal reactions of a CMP containing Inp and azidoproline residues further highlights the versatility of the new isonitrile-containing amino acids.

Synthesis and cellular effects of a mitochondria-targeted inhibitor of the two-pore potassium channel TASK-3.

The two-pore potassium channel TASK-3 has been shown to localize to both the plasma membrane and the mitochondrial inner membrane. TASK-3 is highly expressed in melanoma and breast cancer cells and has been proposed to promote tumor formation. Here we investigated whether pharmacological modulation of TASK-3, and specifically of mitochondrial TASK-3 (mitoTASK-3), had any effect on cancer cell survival and mitochondrial physiology. A novel, mitochondriotropic version of the specific TASK-3 inhibitor IN-THPP has been synthesized by addition of a positively charged triphenylphosphonium moiety. While IN-THPP was unable to induce apoptosis, mitoIN-THPP decreased survival of breast cancer cells and efficiently killed melanoma lines, which we show to express mitoTASK-3. Cell death was accompanied by mitochondrial membrane depolarization and fragmentation of the mitochondrial network, suggesting a role of the channel in the maintenance of the correct function of this organelle. In accordance, cells treated with mitoIN-THPP became rapidly depleted of mitochondrial ATP which resulted in activation of the AMP-dependent kinase AMPK. Importantly, cell survival was not affected in mouse embryonic fibroblasts and the effect of mitoIN-THPP was less pronounced in human melanoma cells stably knocked down for TASK-3 expression, indicating a certain degree of selectivity of the drug both for pathological cells and for the channel. In addition, mitoIN-THPP inhibited cancer cell migration to a higher extent than IN-THPP in two melanoma cell lines. In summary, our results point to the importance of mitoTASK-3 for melanoma cell survival and migration.

A lysyl oxidase-responsive collagen peptide illuminates collagen remodeling in wound healing.

Tissue repair and fibrosis involve the dynamic remodeling of collagen, and accurate detection of these sites is of utmost importance. Here, we use a collagen peptide sensor (1) to visualize collagen formation and remodeling during wound healing in mice and humans. We show that the probe binds selectively to sites of collagen formation and remodeling at different stages of healing. Compared to conventional methods, the peptide sensor localizes preferentially to areas of collagen synthesis and remodeling at the wound edge and not in matured fibrillar collagen. We also demonstrate its applicability for in vivo wound imaging and for discerning differential remodeling in wounds of transgenic mice with altered collagen dynamics. Our findings show the value of 1 as a diagnostic tool to rapidly identify the sites of matrix remodeling in tissue sections, which will aid in the conception of new therapeutic strategies for fibrotic disorders and defective tissue repair.

Evaluating the effects of fluorine on biological properties and metabolic stability of some antitubulin 3-substituted 7-phenyl-pyrroloquinolinones.

A small number of fluorinated 7-phenyl-pyrroloquinolinone (7-PPyQ) derivatives was synthesized in an attempt to improve the metabolic stability of 3N-ethyl-7-PPyQ and 3N-benzoyl-7-PPyQ. The possible impacts of the fluorine-hydrogen isosterism on both biological activity and metabolic stability were evaluated. Introduction of a fluorine atom in the 2 or 3 position of the 7-phenyl ring yielded the 7-PPyQ derivatives 12, 13 and 15, which showed potent cytotoxicity (low micromolar and sub-nanomolar GI50s) both in human leukemic and solid tumor cell lines. None of them induced significant cell death in quiescent and proliferating human lymphocytes. Moreover, 12, 13 and 15 exhibited remarkable cytotoxic activity in the multidrug-resistant cell line CEMVbl100, suggesting that they are not substrates for P-glycoprotein. All compounds inhibited tubulin assembly and the binding of [3H]colchicine to tubulin, with the best activity occurring with compound 15. Mechanistic studies carried out on compound 12 indicated that it caused (a) a strong G2/M arrest; (b) apoptosis in a time- and concentration-dependent manner; (c) a significant production of ROS (in good agreement with the observed mitochondrial depolarization); (d) caspase-3 and poly (ADP-ribose) polymerase activation; and (e) a decrease in the expression of anti-apoptotic proteins. In vivo experiments in a murine syngeneic tumor model demonstrated that compounds 12 and 15 significantly reduced tumor mass at doses four times lower than that required for the reference compound combretastatin A-4 phosphate. Neither monofluorination of the 7-phenyl ring of 3N-ethyl-7-PPyQ nor replacement of the benzoyl function of 3N-benzoyl-7-PPyQ with a 2-fluorobenzoyl moiety led to any improvement in the metabolic stability.