Metformin (Met) is an approved antidiabetic drug currently being explored for repurposing in cancer treatment based on recent evidence of its apparent chemopreventive properties. Met is weakly cationic and targets the mitochondria to induce cytotoxic effects in tumor cells, albeit not very effectively. We hypothesized that increasing its mitochondria-targeting potential by attaching a positively-charged lipophilic substituent would enhance the antitumor activity of Met. In pursuit of this question, we synthesized a set of mitochondria-targeted Met analogs (Mito-Mets) with varying alkyl chain lengths containing a triphenylphosphonium cation (TPP+). In particular, the analog Mito-Met10, synthesized by attaching TPP+ to Met via a 10-carbon aliphatic side chain, was nearly 1,000 times more efficacious than Met at inhibiting cell proliferation in pancreatic ductal adenocarcinoma (PDAC). Notably, in PDAC cells Mito-Met10 potently inhibited mitochondrial complex I, stimulating superoxide and AMPK activation, but had no effect in non-transformed control cells. Moreover, Mito-Met10 potently triggered G1 cell cycle phase arrest in PDAC cells, enhanced their radiosensitivity and more potently abrogated PDAC growth in preclinical mouse models, compared to Met. Collectively, our findings show how improving the mitochondrial targeting of Met enhances its anticancer activities, including in aggressive cancers like PDAC in great need of more effective therapeutic options.

Mutations in the KRAS proto-oncogene are present in 50% of all colorectal cancers and are increasingly associated with chemotherapeutic resistance to frontline biologic drugs. Accumulating evidence indicates key roles for overactive KRAS mutations in the metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis in cancer cells. Here, we sought to exploit the more negative membrane potential of cancer cell mitochondria as an untapped avenue for interfering with energy metabolism in KRAS variant-containing and KRAS WT colorectal cancer cells. Mitochondrial function, intracellular ATP levels, cellular uptake, energy sensor signaling, and functional effects on cancer cell proliferation were assayed. 3-Carboxyl proxyl nitroxide (Mito-CP) and Mito-Metformin, two mitochondria-targeted compounds, depleted intracellular ATP levels and persistently inhibited ATP-linked oxygen consumption in both KRAS WT and KRAS variant-containing colon cancer cells and had only limited effects on nontransformed intestinal epithelial cells. These anti-proliferative effects reflected the activation of AMP-activated protein kinase (AMPK) and the phosphorylation-mediated suppression of the mTOR target ribosomal protein S6 kinase B1 (RPS6KB1 or p70S6K). Moreover, Mito-CP and Mito-Metformin released Unc-51-like autophagy-activating kinase 1 (ULK1) from mTOR-mediated inhibition, affected mitochondrial morphology, and decreased mitochondrial membrane potential, all indicators of mitophagy. Pharmacological inhibition of the AMPK signaling cascade mitigated the anti-proliferative effects of Mito-CP and Mito-Metformin. This is the first demonstration that drugs selectively targeting mitochondria induce mitophagy in cancer cells. Targeting bioenergetic metabolism with mitochondria-targeted drugs to stimulate mitophagy provides an attractive approach for therapeutic intervention in KRAS WT and overactive mutant-expressing colon cancer.

The vaccinia virus DNA polymerase is inherently distributive but acquires processivity by associating with a heterodimeric processivity factor comprised of the viral A20 and D4 proteins. D4 is also an enzymatically active uracil DNA glycosylase (UDG). The presence of an active repair protein as an essential component of the polymerase holoenzyme is a unique feature of the replication machinery. We have shown previously that the A20-UDG complex has a stoichiometry of ϳ1:1, and our data suggest that A20 serves as a bridge between polymerase and UDG. Here we show that conserved hydrophobic residues in the N terminus of A20 are important for its binding to UDG. Our data argue against the assembly of D4 into higher order multimers, suggesting that the processivity factor does not form a toroidal ring around the DNA. Instead, we hypothesize that the intrinsic, processive DNA scanning activity of UDG tethers the holoenzyme to the DNA template. The inclusion of UDG as an essential holoenzyme component suggests that replication and base excision repair may be coupled. Here we show that the DNA polymerase can utilize dUTP as a substrate in vitro. Moreover, uracil moieties incorporated into the nascent strand during holoenzyme-mediated DNA synthesis can be excised by the viral UDG present within this holoenzyme, leaving abasic sites. Finally, we show that the polymerase stalls upon encountering an abasic site in the template strand, indicating that, like many replicative polymerases, the poxviral holoenzyme cannot perform translesion synthesis across an abasic site.The faithful and efficient duplication of genomic DNA is one of the most conserved processes across all forms of life. Although this is a necessary and highly regulated process, it seems that each model organism has evolved unique modifications during this process. Members of the poxvirus family, of which variola virus is the most notable member and vaccinia virus is the experimental prototype, are no exception. Poxviruses are unique in that they complete the replication and maturation of their ϳ200-kb double-stranded DNA genome in the cytoplasm of the infected host cell. This autonomy dictates that poxviruses encode many of the proteins necessary for nucleotide precursor synthesis and metabolism as well as the core set of enzymes and DNA binding proteins that act directly at the replication fork (1, 2). Indeed, genetic, genomic, and biochemical analysis has revealed that eight proteins are responsible for vaccinia virus DNA synthesis and maturation. This repertoire includes the catalytic DNA polymerase (E9 (3-11)), a stoichiometric component of the heterodimeric processivity factor (A20 (12-15)), a second component of the processivity factor (D4) that also possesses uracil DNA glycosylase (UDG) 2 activity (16 -18), a putative superfamily III helicase with known NTPase and DNA primase activity (D5 (19 -23)), a serine/threonine protein kinase (B1 (24 -26)), an abundant phosphoprotein with essential roles in viral replication, transcription, and morphogenesis (H5 ...

Vaccinia virus membrane biogenesis requires the A14 and A17 proteins. We show here that both proteins can associate with membranes co-but not posttranslationally, and we perform a structure function analysis of A14 and A17 using inducible recombinants. In the absence of A14, electron-dense virosomes and distinct clusters of small vesicles accumulate; in the absence of A17, small vesicles form a corona around the virosomes. When the proteins are induced at 12 h postinfection (hpi), crescents appear at the periphery of the electron-dense virosomes, with the accumulated vesicles likely contributing to their formation. A variety of mutant alleles of A14 and A17 were tested for their ability to support virion assembly. For A14, biologically important motifs within the N-terminal or central loop region affected crescent maturation and the immature virion (IV)¡mature virion (MV) transition. For A17, truncation or mutation of the N terminus of A17 engendered a phenotype consistent with the N terminus of A17 recruiting the D13 scaffold protein to nascent membranes. When N-terminal processing was abrogated, virions attempted to undergo the IV-to-MV transition without removing the D13 scaffold and were therefore noninfectious and structurally aberrant. Finally, we show that A17 is phosphorylated exclusively within the C-terminal tail and that this region is a direct substrate of the viral F10 kinase. In vivo, the biological competency of A17 was reduced by mutations that prevented its serine-threonine phosphorylation and restored by phosphomimetic substitutions. Precleavage of the C terminus or abrogation of its phosphorylation diminished the IV¡MV maturation; a block to cleavage spared virion maturation but compromised the yield of infectious virus. P oxviruses are complex DNA viruses that are ubiquitous in nature; the orthopoxvirus family includes variola virus, the etiologic agent of smallpox, and the closely related monkeypox virus, which is endemic in Africa and also causes a severe, acute febrile illness (1, 2). The prototypic poxvirus for experimental study is the closely related vaccinia virus, which has played an eminent role in public health as the vaccine strain used in the successful eradication of smallpox. Poxviruses are unique among DNA viruses in that they replicate exclusively in the cytoplasm of infected cells (2). This physical autonomy is enabled by the presence of ϳ200 genes in the viral genome that encode the machinery for virion entry, gene expression, genome replication and maturation, virion assembly and maturation, and virion egress. The assembly of nascent virions is one of the most unique facets of poxviral infection (3). Unlike most other enveloped viruses, vaccinia virus does not acquire its delimiting membrane by budding into an organelle or through the plasma membrane. Instead, the membrane develops within the cytoplasm, seeded by small patches of membrane that grow into characteristic crescents. As these nascent membrane sheets develop, they engulf virosomal proteins destined for encapsidat...

Temperature-sensitive (ts) mutants of vaccinia virus defective in the B1 kinase demonstrate a conditionally lethal defect in DNA synthesis. B1 is the prototypic member of a new family of protein kinases (vaccinia virus-related kinases, or VRK) that possess distinctive B1-like sequence features within their catalytic motifs (R. J. Nichols and P. Traktman, J. Biol. Chem., in press). Given the striking sequence similarity between B1 and the VRK enzymes, we proposed that they might share overlapping substrate specificity. We therefore sought to determine whether the human and mouse VRK1 enzymes (hVRK1 and mVRK1, respectively) could complement a B1 deficiency in vivo. Recombinant ts2 viruses expressing hVRK1, mVRK1, or wild-type B1 were able to synthesize viral DNA at high temperature, but those expressing the more distantly related human casein kinase 1␣2 could not. Complementation required the enzymatic activity of hVRK1, since a catalytically inactive allele of hVRK1 was unable to confer a temperature-insensitive phenotype. Interestingly, rescue of viral DNA synthesis was not coupled to the ability to phosphorylate H5, the only virus-encoded protein shown to be a B1 substrate in vivo. Expression of hVRK1 during nonpermissive ts2 infections restored virus production and plaque formation, whereas expression of mVRK1 resulted in an intermediate level of rescue. Taken together, these observations indicate that enzymatically active cellular VRK1 kinases can perform the function(s) of B1 required for genome replication, most likely due to overlapping specificity for cellular and/or viral substrates.Vaccinia virus, the prototypic member of the Poxvirus family, encodes a 192-kb double-stranded DNA genome that encodes ϳ200 proteins. This expansive coding capacity enables vaccinia virus to replicate in the cytoplasm of host cells with significant autonomy from cellular factors within the nucleus. A requirement for this compartmentalization is that vaccinia virus must encode and synthesize the majority of components required for productive viral infection, including those involved in transcription and genome replication. Through genetic and biochemical analysis of complementation groups of temperaturesensitive (ts) mutants, five viral open reading frames (ORFs) essential for genome replication have been described. These include the viral DNA polymerase (E9) (6, 7, 26-28, 38, 40, 41, 44), a component of the processive DNA polymerase complex (A20) (17, 32), a DNA-independent nucleotide triphosphatase (D5) (10, 11), uracil DNA glycosylase (D4) (39), and a serine/ threonine protein kinase (B1) (33,34,43). B1 is an essential 34-kDa serine/threonine protein kinase which is made at early times postinfection, prior to the onset of viral DNA synthesis. Phenotypic analysis of two ts mutants with defined lesions in the B1 ORF (ts2 and ts25) has demonstrated that, in the absence of B1, the virus is deficient in its ability to synthesize viral DNA at high temperature (33). The severity of this defect varies with cell type and multiplici...