The abilities of 18 synthetic peptides to target a carrier protein to the nucleus following microinjection into the cytoplasm of HeLa cells were determined. Eight of the sequences chosen for synthesis were based on published nuclear targeting regions as determined by gene fusion and deletion experiments. Six of these sequences were found to be effective when mimicked by a synthetic peptide and conjugated to a carrier protein.One additional peptide was based on a region of lamin L,, a nuclear protein from Xenopus laevis, in which the nuclear targeting region had not been previously investigated. This peptide was also able to target a carrier protein to the nucleus. Eight other peptides which resemble the known targeting signals had little or no nuclear targeting ability. Peptides which were able to target a carrier protein to the nucleus did so within 45 min of injection into the cytoplasm. Two peptides with little or no apparent nuclear targeting ability after 45 min were examined for longer times as well. No increase in nuclear accumulation was observed between 45 min and 4 h after cytoplasmic injection. Comparison of the sequences which were effective at nuclear targeting with those that were not revealed a possible consensus sequence for peptide-mediated nuclear transport.Proteins destined for the nucleus in eucaryotic cells enter through large pore structures distributed evenly over the nuclear envelope. Entry An alternative to genetic manipulation has been chemical synthesis of a peptide which mimics a putative nuclear targeting region. The nuclear targeting sequence of the simian virus 40 (SV40) T antigen has been synthesized and cross-linked to a variety of nonnuclear carrier proteins, such as serum albumin, immunoglobulin G, and ferritin. These conjugates are translocated to the nucleus following microinjection into the cytoplasm of cells (6,14). Changing one amino acid in the sequence (lysine 128 to either threonine or asparagine) yields a dramatic reduction in the degree of transport of the peptide conjugate into nuclei (6, 14). The results of these peptide "'mutations" are consistent with results obtained by mutational analysis of the T antigen itself (10, 13).We synthesized 2 SV40 T-antigen nuclear targeting peptides and 16 others to determine the general utility of peptide-mediated translocation and to define further the sequence requirements for nuclear transport. We found that some but not all of the genetically defined transport sequences could be mimicked by peptide conjugates. Although all of the peptides capable of directing nuclear transport contain a cluster of basic residues, this in itself is not * Corresponding author. sufficient for transport. Most of the nontargeting peptides also contain either three or four basic residues clustered in a short sequence. A comparison of the amino acid sequences of nuclear targeting versus nontargeting peptides has led us to propose a consensus sequence for such peptides. MATERIALS AND METHODSPeptide synthesis. All of the peptides listed in Table 1 wer...

A series of 9-anilinoacridines have been prepared and evaluated for their activity against a multidrug-resistant K1 strain of the malaria parasite Plasmodium falciparum in erythrocyte suspensions. 3,6-Diamino substitution on the acridine ring resulted in lower mammalian cell cytotoxicity and higher antiparasitic activity than other substitution patterns, providing compounds with the highest in vitro therapeutic indices. A new synthesis of 3,6-diamino-9-anilinoacridines, via reduction of the corresponding diazides, gives much higher yields than traditional methods. Within the subset of 3,6-diamino-9-anilinoacridines, there was considerable tolerance to substitution at the 1'-anilino position. In a sharp divergence with structure-activity relationships for high mammalian cell toxicity and anticancer effects, derivatives bearing electron-withdrawing 1'-substituents (e.g., SO2-NHR and CONHR) showed the most potent antimalarial activity (IC50 values of 10-20 nM). Representative compounds were shown to be potent inhibitors of the DNA strand-passing activity of human topoisomerase II and of the DNA decatenation activity of the corresponding parasite enzyme. The 1'-SO2NH2derivative 7n completely inhibited strand passage by Jurkat topoisomerase II at 20 microM, and an increase in linear DNA (indicative of inhibition of religation) was seen at or above 1 microM. It also inhibited the decatenating activity of the parasite topoisomerase II at 6 microM and above. In contrast, the analogous compound without the 3,6-diamino substituent was inactive in both assays up to 100 microM. Overall, there was a positive relationship between the ability of the drugs to inhibit parasite growth in culture and their ability to inhibit parasite topoisomerase II activity in an isolated enzyme assay. The 1'-SO2NH2 derivative 7n showed a high IVTI (1000) and was a potent inhibitor of both P. falciparum in vitro (IC50 20 nM) and P. falciparum-derived topoisomerase II. However, the compound was inactive against Plasmodium berghei in mice; reasons may include rapid metabolic inactivation (possibly by N-acetylation) and/or poor distribution.

Members of the class of 9-anilinoacridine topoisomerase II inhibitors bearing lipophilic electron-donating 1'-anilino substituents are active against both the promastigote and amastigote forms of the parasite Leishmania major. A series of analogues of the known 1'-NHhexyl lead compound were prepared and evaluated against L. major in macrophage culture to further develop structure-activity relationships (SAR). Toxicity toward mammalian cells was measured in a human leukemia cell line, and the ratio of the two IC50 values (IC50(J)/IC50(L)) was used as a measure of the in vitro therapeutic index (IVTI). A 3,6-diNMe2 substitution pattern on the acridine greatly increased toxicity to L. major without altering mammalian toxicity, increasing IVTIs over that of the lead compound. The 2-OMe, 6-Cl acridine substitution pattern used in the antimalarial drug mepacrine also resulted in potent antileishmanial activity and high IVTIs. Earlier suggestions of the utility of 2'-OR groups in lowering mammalian cytotoxicity were not borne out in this wider study. A series of very lipophilic 1'-NRR (symmetric dialkylamino)-substituted analogues showed relatively high antileishmanial potency, but no clear trend was apparent across the series, and none were superior to the 1'-NH(CH2)5Me subclass. Subsets of the most active 1'-N(R)(CH2)5Me- and 1'-N(alkyl)2-substituted compounds against L. major were also evaluated against Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei, but no consistent SAR could be discerned in these physiologically diverse test systems. The present study has confirmed earlier conclusions that lipophilic electron-donating groups at the 1'-position of 9-anilinoacridines provide high activity against L. major, but the SAR patterns observed do not carry over to the other parasites studied.

Tumor metastases that impede the function of vital organs are a major cause of cancer related mortality. Mitochondrial oxidative stress induced by hypoxia, low nutrient levels, or other stresses, such as genotoxic events, act as key drivers of the malignant changes in primary tumors to enhance their progression to metastasis. Emerging evidence now indicates that mitochondrial modifications and mutations resulting from oxidative stress, and leading to OxPhos stimulation and/or enhanced reactive oxygen species (ROS) production, are essential for promoting and sustaining the highly metastatic phenotype. Moreover, the modified mitochondria in emerging or existing metastatic cancer cells, by their irreversible differences, provide opportunities for selectively targeting their mitochondrial functions with a one-two punch. The first blow would block their anti-oxidative defense, followed by the knockout blow—promoting production of excess ROS, capitulating the terminal stage—activation of the mitochondrial permeability transition pore (mPTP), specifically killing metastatic cancer cells or their precursors. This review links a wide area of research relevant to cellular mechanisms that affect mitochondria activity as a major source of ROS production driving the pro-oxidative state in metastatic cancer cells. Each of the important aspects affecting mitochondrial function are discussed including: hypoxia, HIFs and PGC1 induced metabolic changes, increased ROS production to induce a more pro-oxidative state with reduced antioxidant defenses. It then focuses on how the mitochondria, as a major source of ROS in metastatic cancer cells driving the pro-oxidative state of malignancy enables targeting drugs affecting many of these altered processes and why the NSAIDs are an excellent example of mitochondria-targeted agents that provide a one-two knockout activating the mPTP and their efficacy as selective anticancer metastasis drugs.

Antimalarial 9-anilinoacridines are potent inhibitors of parasite DNA topoisomerase II both in vitro and in situ. 3,6-Diamino substitution on the acridine ring greatly improves parasiticidal activity against Plasmodium falciparum by targeting DNA topoisomerase II. A series of 9-anilinoacridines were investigated for their abilities to inhibit ␤-hematin formation, to form drug-hematin complexes, and to enhance hematin-induced lysis of red blood cells. Inhibition of ␤-hematin formation was minimal with 3,6-diamino analogs of 9-anilinoacridine and greatest with analogs with a 3,6-diCl substitution together with an electron-donating group in the 1-anilino position. On the other hand, the presence of a 1-N(CH 3 ) 2 group in the anilino ring produced compounds that strongly inhibited ␤-hematin formation but which did not appear to be sensitive to the nature of the substitutions in the acridine nucleus. The derivatives bound hematin, and Job's plots of UV-visible absorbance changes in drug-hematin complexes at various molar ratios indicated a stoichiometric ratio of 1:2. The drugs enhanced hematin-induced red blood cell lysis at low concentrations (<4 M). These studies open up the novel possibility of development of 9-anilinoacridine antimalarials that target not only DNA topoisomerase II but also ␤-hematin formation, which should help delay the rapid onset of resistance to drugs acting at only a single site.