ABSTRACT
Activating the intrinsic apoptosis pathway with small molecules is now a clinically validated approach to cancer therapy. In contrast, blocking apoptosis to prevent the death of healthy cells in disease settings has not been achieved. Caspases have been favored, but they act too late in apoptosis to provide long-term protection. The critical step in committing a cell to death is activation of BAK or BAX, pro-death BCL-2 proteins mediating mitochondrial damage. Apoptosis cannot proceed in their absence. Here we show that WEHI-9625, a novel tricyclic sulfone small molecule, binds to VDAC2 and promotes its ability to inhibit apoptosis driven by mouse BAK. In contrast to caspase inhibitors, WEHI-9625 blocks apoptosis before mitochondrial damage, preserving cellular function and long-term clonogenic potential. Our findings expand on the key role of VDAC2 in regulating apoptosis and demonstrate that blocking apoptosis at an early stage is both advantageous and pharmacologically tractable.
Subject(s)
Apoptosis/physiology , Small Molecule Libraries/metabolism , Voltage-Dependent Anion Channel 2/physiology , bcl-2 Homologous Antagonist-Killer Protein/physiology , Animals , Mice , Protein Binding , Voltage-Dependent Anion Channel 2/metabolismABSTRACT
Mitochondrial apoptosis is mediated by BAK and BAX, two proteins that induce mitochondrial outer membrane permeabilization, leading to cytochrome c release and activation of apoptotic caspases. In the absence of active caspases, mitochondrial DNA (mtDNA) triggers the innate immune cGAS/STING pathway, causing dying cells to secrete type I interferon. How cGAS gains access to mtDNA remains unclear. We used live-cell lattice light-sheet microscopy to examine the mitochondrial network in mouse embryonic fibroblasts. We found that after BAK/BAX activation and cytochrome c loss, the mitochondrial network broke down and large BAK/BAX pores appeared in the outer membrane. These BAK/BAX macropores allowed the inner mitochondrial membrane to herniate into the cytosol, carrying with it mitochondrial matrix components, including the mitochondrial genome. Apoptotic caspases did not prevent herniation but dismantled the dying cell to suppress mtDNA-induced innate immune signaling.
Subject(s)
Apoptosis , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/metabolism , Animals , Cytochromes c/metabolism , DNA, Mitochondrial/metabolism , Fibroblasts , Gene Knockout Techniques , HeLa Cells , Humans , Mice , Mice, Inbred C57BL , Mitochondrial Membranes/chemistry , Protein Multimerization , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2-Associated X Protein/geneticsABSTRACT
The impact of PEGylation on the pharmacokinetics and biodistribution of (3)H-labeled poly l-lysine dendrimers has been investigated after intravenous administration to rats. The volumes of distribution, clearance and consequently the plasma half-lives of the PEGylated dendrimers were markedly dependent on the total molecular weight of the PEGylated dendrimer, but were not specifically affected by the PEG chain length alone. In general, the larger dendrimer constructs (i.e. >30 kDa) had reduced volumes of distribution, were poorly renally cleared and exhibited extended elimination half-lives ( t 1/2 1-3 days) when compared to the smaller dendrimers (i.e. <20 kDa) which were rapidly cleared from the plasma principally into the urine ( t 1/2 1-10 h). At later time points the larger dendrimers concentrated in the organs of the reticuloendothelial system (liver and spleen); however, the absolute extent of accumulation was low. Size exclusion chromatography of plasma and urine samples revealed that the PEGylated dendrimers were considerably more resistant to biodegradation in vivo than the underivatized poly l-lysine dendrimer cores. The results suggest that the size of PEGylated poly l-lysine dendrimer complexes can be manipulated to optimally dictate their pharmacokinetics, biodegradation and bioresorption behavior.
Subject(s)
Dendrimers/pharmacokinetics , Polyethylene Glycols/pharmacokinetics , Polylysine/pharmacokinetics , Animals , Biological Availability , Dendrimers/administration & dosage , Dendrimers/chemical synthesis , Drug Delivery Systems/methods , Half-Life , Injections, Intravenous , Male , Metabolic Clearance Rate , Molecular Structure , Molecular Weight , Polylysine/administration & dosage , Polylysine/blood , Polylysine/urine , Rats , Rats, Sprague-Dawley , Tissue Distribution , TritiumABSTRACT
Cationic poly-L-lysine 3H-dendrimers with either 16 or 32 surface amine groups (BHALys [Lys]4 [3H-Lys]8 [NH2]16 and BHALys [Lys]8 [3H-Lys]16 [NH2]32, generation 3 and 4, respectively) have been synthesized and their pharmacokinetics and biodistribution investigated after intravenous administration to rats. The species in plasma with which radiolabel was associated was also investigated by size exclusion chromatography (SEC). Rapid initial removal of radiolabel from plasma was evident for both dendrimers (t(1/2) < 5 min). Approximately 1 h postdose, however, radiolabel reappeared in plasma in the form of free lysine and larger (but nondendrimer) species that coeluted with albumin by SEC. Plasma and whole blood pharmacokinetics were similar, precluding interaction with blood components as a causative factor in either the rapid removal or reappearance of radioactivity in plasma. Administration of monomeric 3H L-lysine also resulted in the appearance in plasma of a radiolabeled macromolecular species that coeluted with albumin by SEC, suggesting that biodegradation of the dendrimer to L-lysine and subsequent bioresorption may explain the pharmacokinetic profiles. Capping the Lys8 dendrimer with D-lysine to form BHALys [Lys]4 [3H-Lys]8 [D-Lys]16 [NH2]32 resulted in similar, and very rapid, initial disappearance kinetics from plasma when compared to the L-lysine capped dendrimer. Since significant extravasation of these large hydrophilic molecules seems unlikely, this most likely reflects both elimination and extensive binding to vascular surfaces. Capping with "non-natural" D-lysine also appeared to render the dendrimer essentially inert to the biodegradation process. For the L-lysine capped dendrimers, radiolabel was widely distributed throughout the major organs, with no apparent selectivity for organs of the reticuloendothelial system. In contrast, a greater proportion of the administered radiolabel was recovered in the organs of the reticuloendothelial system for the D-lysine capped system, as might be expected for a nondegrading circulating foreign colloid. To our knowledge this is the first data to demonstrate the biodegradation/bioresorption of poly-L-lysine dendrimers and has significant implications for the utility of these systems as drugs or drug delivery systems.