Comparison of potency of peptide-derived caspase inhibitors in cellular lamin cleavage assay, enzymatic activity and cell permeability.

<p>nd = not determined</p><p>a = z-VEID-TFPM enzymatic IC₅₀ is less than 0.0017 due to limit of enzymatic assay detection.</p><p>*Enzymatic IC₅₀ values were determined after a 15 minute enzyme/inhibitor preincubation and 40 minute enzyme reaction.</p

Effect of peptide-based caspase inhibitors on SKNAS cells as determined using the Caspase-Glo® 6 assay.

<p>SKNAS cells were treated with Ac-VEID-CHO (•) or Ac-DEVD-CHO (▪) prior to addition of 3 µM staurosporine for 6 hours and detection of VEID-ase activity as described in Experimental Procedures. Concentration inhibition curves were performed in duplicate and represent 1 of at least 3 experiments with similar results. Concentration-response curves for each inhibitor were normalized to zero and 100% based on no staurosporine or DMSO, respectively. The mean and standard error of the mean are reported.</p

Effect of peptide-based caspase inhibitors on the generation of cleaved lamin A/C in SKNAS cells.

<p>(A) SKNAS cells were treated with z-VEID-FMK (♦), z-DEVD-FMK (▪), Q-VD-OPh (•), Ac-VEID-CHO (▴) or Ac-DEVD-CHO (○) prior to addition of 3 µM staurosporine for 6 hours. (B) SKNAS cells were treated with z-ID-TFPM (•), z-EID-TFPM (▴) or z-VEID-TFPM (▪) prior to addition of 3 µM staurosporine for 6 hours. Detection of the small lamin A/C cleavage product was performed as described in Experimental Procedures. Concentration inhibition curves were performed in duplicate and represent 1 of at least 3 experiments with similar results. Concentration-response curves for each inhibitor were normalized to zero and 100% based on no staurosporine or DMSO, respectively. The mean and standard error of the mean are reported.</p

Potency of peptide-derived caspase inhibitors possessing aldehyde (CHO) and fluoromethyl ketone (FMK) warheads against executioner caspases.

<p>*IC₅₀ values were determined after a 15 minute enzyme/inhibitor preincubation and 40 minute enzyme reaction. Due to the capacity for time-dependent inhibition with irreversible inhibitors the values reported can be considered “apparent IC₅₀”.</p

Effect of staurosporine on the generation of cleaved lamin A/C in SKNAS cells.

<p>SKNAS cells were treated with the indicated concentration of staurosporine for 6 hours prior to detection of the small lamin A/C cleavage product as described in Experimental Procedures. The assay was performed in triplicate one time. The mean and standard error of the mean are reported.</p

Western blot detection of recombinant GST-lamin A processing by purified caspases.

<p>(A) The indicated concentration of caspase-6 was incubated with GST-lamin A for two hours. (B) 300 Units of caspases 1–9 were incubated with GST-lamin A for two hours. Intact and cleaved lamin A were detected via western blotting using anti-GST antibody.</p

Effect of staurosporine on the release of Lactate Dehydrogenase in SKNAS cells.

<p>SKNAS cells were treated with the indicated concentration of staurosporine for 2 (•), 4 (▪), 6 (▴) or 8 (♦) hours prior to detection of LDH release to the cell supernatant. The assay was performed in triplicate and represents 1 of at least 2 experiments with similar results. The data was normalized to fold increase over DMSO treatment. The mean and standard error of the mean are reported.</p

Apoptosis-mediated cleavage of lamin A/C is elevated in wild-type relative to caspase-6 KO fibroblasts.

<p>Fibroblasts derived from caspase-6 KO (▪) or wild type (•) mice were treated with the indicated concentration of staurosporine for 6 hours prior to detection of the small lamin A/C cleavage product. The assay was performed in quadruplicate two times with similar results; mean and standard error of the mean are reported.</p

Western blot detection of lamin A/C from SKNAS neuroblastoma cells upon staurosporine treatment.

<p>(A) Schematic of the N- and C-terminal globular domains of Lamin A/C with VEID-containing central α-helical region as the site of caspase-6 proteolysis. (B) SKNAS cells were treated with DMSO control or staurosporine for 6 hours prior to cell lysis. Lysates were probed for small lamin A/C subunit (Lanes 1–2), large lamin A/C subunit (Lanes 3–4) or total lamin A/C (Lanes 5–6). (C) SKNAS cells were treated with DMSO or staurosporine for the indicated time prior to cell lysis. Lysates were probed for large lamin A/C subunit (red) or β-Actin (green).</p

Lipid Tales: Optimizing Arylomycin Membrane Anchors

Multidrug-resistant bacteria are spreading at alarming rates, and despite extensive efforts, no new antibiotic class with activity against Gram-negative bacteria has been approved in over 50 years. LepB inhibitors (LepBi) based on the arylomycin class of natural products are a novel class of antibiotics and function by inhibiting the bacterial type I signal peptidase (SPase) in Gram-negative bacteria. One critical aspect of LepBi development involves optimization of the membrane-anchored lipophilic portion of the molecule. We therefore developed an approach that assesses the effect of this portion on the complicated equilibria of plasma protein binding, crossing the outer membrane of Gram-negative bacteria and anchoring in the bacterial inner membrane to facilitate SPase binding. Our findings provide important insights into the development of antibacterial agents where the target is associated with the inner membrane of Gram-negative bacteria