8 research outputs found
Investigating the mechanism of cytotoxic lymphocyte resistance to perforin
© 2020 Jesse Alexander Rudd-SchmidtCytotoxic lymphocytes are highly efficient killer cells of the immune system. They destroy cognate target cells by secreting highly toxic effector molecules, the pore-forming protein perforin and pro-apoptotic serine proteases granzymes, into the confines of the immune synapse. Despite both the lymphocyte and target cell plasma membrane being equally exposed to the perforin and granzymes, the lymphocytes invariably survive that encounter as they remain resistant to perforin pores. This project investigates the mechanisms behind this unique phenomenon
Labeling phospholipid membranes with lipid mimetic luminescent metal complexes
AbstractLipid-mimetic metallosurfactant based luminophores are promising candidates for labeling phospholipid membranes without altering their biophysical characteristics. The metallosurfactants studied exhibit high structural and physicochemical similarity to phospholipid molecules, designed to incorporate into the membrane structure without the need for covalent attachment to a lipid molecule. In this work, two lipid-mimetic phosphorescent metal complexes are described: [Ru(bpy)2(dn-bpy)]2+ and [Ir(ppy)2(dn-bpy)]+ where bpy is 2,2′-bipyridine, dn-bpy is 4,4′-dinonyl-2,2′-bipyridine and ppy is 2-phenylpyridine. Apart from being lipid-mimetic in size, shape and physical properties, both complexes exhibit intense photoluminescence and enhanced photostability compared with conventional organic fluorophores, allowing for prolonged observation. Moreover, the large Stokes shift and long luminescence lifetime associated with these complexes make them more suitable for spectroscopic studies. The complexes are easily incorporated into dimyristoil-phosphatidyl-choline (DMPC) liposomes by mixing in the organic solvent phase. DLS reveals the labeled membranes form liposomes of similar size to that of neat DMPC membrane. Synchrotron Small-Angle X-ray Scattering (SAXS) measurements confirmed that up to 5% of either complex could be incorporated into DMPC membranes without producing any structural changes in the membrane. Fluorescence microscopy reveals that 0.5% label content is sufficient for imaging. Atomic Force Microscopic imaging confirms that liposomes of the labeled bilayers on a mica surface can fuse into a flat lamellar membrane that is morphologically identical to neat lipid membranes. These results demonstrate the potential of such lipid-mimetic luminescent metal complexes as a new class of labels for imaging lipid membranes
Exploration of a series of 5-arylidene-2-thioxoimidazolidin-4-ones as inhibitors of the cytolytic protein perforin
A series of novel 5-arylidene-2-thioxoimidazolidin-4-ones were investigated as inhibitors of the lymphocyte-expressed pore-forming protein perform. Structure activity relationships were explored through variation of an isoindolinone or 3,4-dihydroisoquinolinone subunit on a fixed 2-thioxoimidazolidin-4-one/thiophene core. The ability of the resulting compounds to inhibit the lytic activity of both isolated perform protein and perforin delivered in situ by natural killer cells was determined. A number of compounds showed excellent activity at concentrations that were nontoxic to the killer cells, and several were a significant improvement on previous classes of inhibitors, being substantially more potent and soluble. Representative examples showed rapid and reversible binding to immobilized mouse perforin at low concentrations
Exploration of a series of 5-arylidene-2-thioxoimidazolidin-4-ones as inhibitors of the cytolytic protein perforin
A series of novel 5-arylidene-2-thioxoimidazolidin-4-ones were investigated as inhibitors of the lymphocyte-expressed pore-forming protein perform. Structure activity relationships were explored through variation of an isoindolinone or 3,4-dihydroisoquinolinone subunit on a fixed 2-thioxoimidazolidin-4-one/thiophene core. The ability of the resulting compounds to inhibit the lytic activity of both isolated perform protein and perforin delivered in situ by natural killer cells was determined. A number of compounds showed excellent activity at concentrations that were nontoxic to the killer cells, and several were a significant improvement on previous classes of inhibitors, being substantially more potent and soluble. Representative examples showed rapid and reversible binding to immobilized mouse perforin at low concentrations
Exploration of a Series of 5‑Arylidene-2-thioxoimidazolidin-4-ones as Inhibitors of the Cytolytic Protein Perforin
A series of novel 5-arylidene-2-thioxoimidazolidin-4-ones
were
investigated as inhibitors of the lymphocyte-expressed pore-forming
protein perforin. Structure–activity relationships were explored
through variation of an isoindolinone or 3,4-dihydroisoquinolinone
subunit on a fixed 2-thioxoimidazolidin-4-one/thiophene core. The
ability of the resulting compounds to inhibit the lytic activity of
both isolated perforin protein and perforin delivered in situ by natural
killer cells was determined. A number of compounds showed excellent
activity at concentrations that were nontoxic to the killer cells,
and several were a significant improvement on previous classes of
inhibitors, being substantially more potent and soluble. Representative
examples showed rapid and reversible binding to immobilized mouse
perforin at low concentrations (≤2.5 μM) by surface plasmon
resonance and prevented formation of perforin pores in target cells
despite effective target cell engagement, as determined by calcium
influx studies. Mouse PK studies of two analogues showed <i>T</i><sub>1/2</sub> values of 1.1–1.2 h (dose of 5 mg/kg iv) and
MTDs of 60–80 mg/kg (ip)