Agents that chemically modify DNA form a backbone of many cancer treatments. A key problem for DNA modifying agents is lack of specificity. To address this issue, we designed novel molecular scaffolds, termed An-Hq and An-Hq2, which are activated by a hallmark of some cancers: elevated concentrations of reactive oxygen species. Elevated reactive oxygen species are linked to oncogenesis and is found to increase in several aggressive cancers. The agents are quinones that, upon oxidation, form highly electrophilic species. In vitro studies identified the mode of addition to DNA. The aniline portion of An-Hq serves to enhance nucleophilic addition to the ethyl phenyl ether instead of forming common Michael additions. Structural characterization showed the agents add to 2′-deoxyguanosine at the N2,N3-positions. The product formed is a bulky hydroxy-N2,3-benzetheno-2′-deoxyguanosine adduct. In addition, the oxidatively activated agents added to 2′-deoxyadenosine and 2′-deoxycytidine, but not thymidine or 2′-deoxyinosine. These findings are confirmed by primer extension analysis of a 392 base pair DNA. The full-length primer extension product was reduced by 69.0 ± 0.6% upon oxidative activation of An-Hq2 compared to controls. Little sequence dependence was observed with 76% of guanine, adenine, and cytosine residues showing an increase in extension stops between two and four fold above controls. Benzetheno-nucleobase addition to double stranded DNA was confirmed by LC/MS of a self-complementary oligonucletide. Experiments were carried out to confirm in vivo DNA damage. Because of the lesion identified in vitro, we reasoned that nucleotide excision repair should be involved in reversing the effects of these oxidatively activated agents and enhance toxicity in Drosophila melanogaster. Using an RNAi based approach, Ercc1 was silenced and survival monitored after injection of an agent. As expected, bulky cross-linking DNA modifying agents, cisplatin and chlorambucil, showed statistically significant enhanced toxicity in Drosophila with silenced Ercc1. In addition, 5-fluorouracil, which does not produce bulky lesions, showed no selective toxicity. An-Hq and An-Hq2 showed statistically significant toxicity in Drosophila with silenced Ercc1. Examination of cytotoxicity shows renal carcinoma cell lines as a target of these agents with a median IC50 of 1.8 μM. Taken together, this data shows that the designed oxidatively-activated agents form distinct, bulky DNA modifications that prove difficult for cancer cells possessing an elevated reactive oxygen species phenotype to overcome. The modification produced is relatively unique among anticancer agents.
This study explores the possible use of reactive oxygen-activated DNA modifying agents against acute myeloid leukemia (AML). A key amine on the lead agent was investigated via cytotoxicity assays and was found necessary for potency. The two best compounds were screened via the NCI-60 cell panel. These two compounds had potency between 200 and 800 nM against many of the leukemia cancer cell types. Subsequent experiments explored activity against a transformed AML model that mimics the molecular signatures identified in primary AML patient samples. A lead compound had an IC50 of 760 nM against this AML cell line as well as a therapeutic index of 7.7 ± 3 between the transformed AML model cell line and non-cancerous human CD34+ blood stem/progenitor cells (UCB). The selectivity was much greater than the mainstays of AML treatment: doxorubicin and cytarabine. This manuscript demonstrates that this novel type of agent may be useful against AML.
Ferrostatin-1 (Fer-1) is a lipophilic antioxidant that effectively blocks ferroptosis, a distinct non-apoptotic form of cell death caused by lipid peroxidation. During many infections, both pathogens and host cells are subjected to oxidative stress, but the occurrence of ferroptosis had not been investigated. We examined ferroptosis in macrophages infected with the pathogenic yeast Histoplasma capsulatum. Unexpectedly, Fer-1 not only reduced the death of macrophages infected in vitro, but inhibited the growth of H. capsulatum and related species Paracoccidioides lutzii and Blastomyces dermatitidis at concentrations under 10 μm. Other antioxidant ferroptosis inhibitors, including liproxstatin-1, did not prevent fungal growth or reduce macrophage death. Structural analysis revealed a potential similarity of Fer-1 to inhibitors of fungal sterol synthesis, and ergosterol content of H. capsulatum decreased more than twofold after incubation with Fer-1. Strikingly, additional Fer-1 analogues with slight differences from Fer-1 had limited impact on fungal growth. In conclusion, the ferroptosis inhibitor Fer-1 has unexpected antifungal potency distinct from its antiferroptotic activity.
There is currently no FDA approved treatment for cocaine abuse. Our laboratory has created a humanized anti‐cocaine monoclonal antibody (mAb), h2E2, that has high affinity and specificity for cocaine and is cleared slowly in rodents. The mAb h2E2 sequesters cocaine in the plasma and prevents cocaine entry into the brain in rodents. The antigen binding portion, the Fab fragment, of h2E2 has the same high affinity and selectivity as its whole mAb counterpart. The Fab fragment should also sequester cocaine into the plasma compartment but is expected to have a more rapid clearance than the intact mAb. This study elucidated the pharmacokinetics of the Fab fragment and its effects on cocaine’s pharmacokinetics in mice. Catheterized mice were injected with Fab (80 mg/kg, i.v., pH=7.4, PBS) (n=8) and blood samples collected from the tip of the tail at designated time points over three days. Fab concentrations in blood samples were quantified using an ELISA assay. Two groups of catheterized mice were injected under mild restraint with Fab (82 mg/kg, i.v. pH= 7.4, PBS, generated by enzymatic digestion of whole mAb h2E2) or vehicle (PBS, pH=7.4). One hour later, mice were injected with an equimolar dose of cocaine HCl (0.56 mg/kg, i.v.). At 8 time points between 0.75 and 60 min, mice were decapitated, and trunk blood was collected. Sodium pentobarbital (50 mg/kg, i.p.) was administered 3 minutes prior to decapitation. For the 0.75 and 1.5‐minute time points, cocaine was injected into anesthetized rats. Blood (typically 0.5–2 mL) was placed into 2‐mL polypropylene tubes and then centrifuged at 5000 g for 3 minutes. Plasma was pipetted into polypropylene microcentrifuge tubes and rapidly frozen on dry ice and stored at −80°C until LC‐MS/MS analysis. This experiment was performed three times. Total cocaine concentrations were quantified using LC‐MS/MS. The pharmacokinetic profile of the Fab fragment in mice was described by a 2‐compartment model (Figure 1). The Fab fragment has a mean distribution half‐life (t1/2α) of 16.7 minutes and a terminal elimination half‐life (t1/2β) of 7.1 hours (Figure 1). In the presence of the Fab fragment, cocaine concentrations in the plasma increased by 4.5‐fold compared to vehicle animals (Figure 2). The initial volume of distribution of cocaine decreased approximately 4.5‐fold in the presence of Fab. The Fab fragment increases the concentration of cocaine in the plasma by approximately 4.5‐fold compared to vehicle controls. Although the data is not shown, it is hypothesized that the Fab fragment, like its mAb counterpart, prevents cocaine from crossing the blood‐brain barrier and entering the brain. With a 24‐fold decrease in elimination half‐life (7.1 hours) compared to the intact h2E2 (7.8 days) and predicted rapid urinary excretion, the Fab fragment may be useful for the treatment of cocaine overdoses. Support or Funding Information This work is supported by the National Institutes of Health, National Institute on Drug Abuse grant number U01DA039550 (to ABN). The pharmacokinetics of t...
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