Xiaohua Peng

Hydrogen Peroxide Inducible DNA Cross-Linking Agents: Targeted Anticancer Prodrugs

The major concern for anticancer chemotherapeutic agents is the host toxicity. The development of anticancer prodrugs targeting the unique biochemical alterations in cancer cells is an attractive approach to achieve therapeutic activity and selectivity. We designed and synthesized a new type of nitrogen mustard prodrug that can be activated by high level of reactive oxygen species (ROS) found in cancer cells to release the active chemotherapy agent. The activation mechanism was determined by NMR analysis. The activity and selectivity of these prodrugs toward ROS was determined by measuring DNA interstrand cross-links and/or DNA alkylations. These compounds showed 60-90% inhibition toward various cancer cells, while normal lymphocytes were not affected. To the best of our knowledge, this is the first example of H(2)O(2)-activated anticancer prodrugs.

ROS‐Inducible DNA Cross‐Linking Agent as a New Anticancer Prodrug Building Block

Some antitumor drugs react with DNA by inducing DNA interstrand cross-links (ICLs) which can block DNA transcription and replication.[1] ICL-inducing agents, such as nitrogen mustard, mitomycin C, cisplatin, and psoralens have been used in cancer therapy.[2] However, the major disadvantage of these agents is their poor selectivity for cancer cells. One novel approach to reduce the toxicity of cross-linking agents for normal cells would be the creation of prodrugs that undergo tumor-specific activation. Inducible DNA cross-linking or alkylating agents have been developed by several research groups.[3-6] However, few of them can induce DNA crosslinks selectively under tumor-specific conditions. One of the exclusive features of cancer cells is the high level of oxidative stress that is associated with the increased amounts of reactive oxygen species (ROS).[7-10] Therefore, it would be advantageous to develop novel cross-linking agents that can be activated by the high level of ROS in cancer cells.

Replication termination mechanism as revealed by Tus-mediated polar arrest of a sliding helicase

The replication terminator protein Tus of Escherichia coli promotes polar fork arrest at sequence-specific replication termini (Ter) by antagonizing DNA unwinding by the replicative helicase DnaB. Here, we report that Tus is also a polar antitranslocase. We have used this activity as a tool to uncouple helicase arrest at a Tus–Ter complex from DNA unwinding and have shown that helicase arrest occurred without the generation of a DNA fork or a bubble of unpaired bases at the Tus–Ter complex. A mutant form of Tus, which reduces DnaB–Tus interaction but not the binding affinity of Tus for Ter DNA, was also defective in arresting a sliding DnaB. A model of polar fork arrest that proposes melting of the Tus–Ter complex and flipping of a conserved C residue of Ter at the blocking but not the nonblocking face has been reported. The model suggests that enhanced stability of Tus–Ter interaction caused by DNA melting and capture of a flipped base by Tus generates polarity strictly by enhanced protein–DNA interaction. In contrast, the observations presented here show that polarity of helicase and fork arrest in vitro is generated by a mechanism that not only involves interaction between the terminator protein and the arrested enzyme but also of Tus with Ter DNA, without any melting and base flipping in the termination complex.

Reactive oxygen species (ROS) inducible DNA cross-linking agents and their effect on cancer cells and normal lymphocytes

Reducing host toxicity is one of the main challenges of cancer chemotherapy. Many tumor cells contain high levels of ROS that make them distinctively different from normal cells. We report a series of ROS-activated aromatic nitrogen mustards that selectively kill chronic lymphocytic leukemia (CLL) over normal lymphocytes. These agents showed powerful DNA cross-linking abilities when coupled with H2O2, one of the most common ROS in cancer cells, whereas little DNA cross-linking was detected without H2O2. Consistent with chemistry observation, in vitro cytotoxicity assay demonstrated that these agents induced 40–80% apoptosis in primary leukemic lymphocytes isolated from CLL patients but less than 25% cell death to normal lymphocytes from healthy donors. The IC50 for the most potent compound (2) was ∼5 μM in CLL cells, while the IC50 was not achieved in normal lymphocytes. Collectively, these data provide utility and selectivity of these agents that will inspire further and effective applications.

Facile SNP detection using bifunctional, cross-linking oligonucleotide probes

A facile, sensitive method for detecting specific sequences of oligonucleotides was developed. Detection of DNA sequences with single nucleotide discrimination is achieved by combining the selectivity of hybridization with an efficient cross-linking reaction. Readily synthesized bifunctional oligonucleotide probes containing a modified pyrimidine that is capable of forming interstrand cross-links under mild oxidative conditions internally, and biotin at their 5′-termini were used to discriminate between 16-nt long sites in plasmid DNA that differ by a single nucleotide. The target sequence was detected via fluorescence spectroscopy by utilizing conjugates of avidin and horseradish peroxidase in a microtiter plate assay. The method is able to detect as little as 250 fmol of target without using PCR and exhibits single nucleotide discrimination that approaches 200:1. In principle, this method is capable of probing any target sequence containing a 2′-deoxyadenosine.

Substituent Effects on Oxidation‐Induced Formation of Quinone Methides from Arylboronic Ester Precursors

A series of arylboronic esters containing different aromatic substituents and various benzylic leaving groups (Br or N(+)Me3Br(-)) have been synthesized. The substituent effects on their reactivity with H2O2 and formation of quinone methide (QM) have been investigated. NMR spectroscopy and ethyl vinyl ether (EVE) trapping experiments were used to determine the reaction mechanism and QM formation, respectively. QMs were not generated during oxidative cleavage of the boronic esters but by subsequent transformation of the phenol products under physiological conditions. The oxidative deboronation is facilitated by electron-withdrawing substituents, such as aromatic F, NO2, or benzylic N(+)Me3Br(-), whereas electron-donating substituents or a better leaving group favor QM generation. Compounds containing an aromatic CH3 or OMe group, or a good leaving group (Br), efficiently generate QMs under physiological conditions. Finally, a quantitative relationship between the structure and activity has been established for the arylboronic esters by using a Hammett plot. The reactivity of the arylboronic acids/esters and the inhibition or facilitation of QM formation can now be predictably adjusted. This adjustment is important as some applications may benefit and others may be limited by QM generation.

Aromatic Nitrogen Mustard‐Based Prodrugs: Activity, Selectivity, and the Mechanism of DNA Cross‐Linking

Three novel H2O2-activated aromatic nitrogen mustard prodrugs (6-8) are reported. These compounds contain a DNA alkylating agent connected to a H2O2-responsive trigger by different electron-withdrawing linkers so that they are inactive towards DNA but can be triggered by H2O2 to release active species. The activity and selectivity of these compounds towards DNA were investigated by measuring DNA interstrand cross-link (ICL) formation in the presence or absence of H2O2. An electron-withdrawing linker unit, such as a quaternary ammonia salt (6), a carboxyamide (7), and a carbonate group (8), is sufficient to deactivate the aromatic nitrogen mustard resulting in less than 1.5 % cross-linking formation. However, H2O2 can restore the activity of the effectors by converting a withdrawing group to a donating group, therefore increasing the cross-linking efficiency (>20 %). The stability and reaction sites of the ICL products were determined, which revealed that alkylation induced by 7 and 8 not only occurred at the purine sites but also at the pyrimidine site. For the first time, we isolated and characterized the monomer adducts formed between the canonical nucleosides and the aromatic nitrogen mustard (15) which supported that nitrogen mustards reacted with dG, dA, and dC. The activation mechanism was studied by NMR spectroscopic analysis. An in vitro cytotoxicity assay demonstrated that compound 7 with a carboxyamide linker dramatically inhibited the growth of various cancer cells with a GI50 of less than 1 μM, whereas compound 6 with a charged linker did not show any obvious toxicity in all cell lines tested. These data indicated that a neutral carboxyamide linker is preferable for developing nitrogen mustard prodrugs. Our results showed that 7 is a potent anticancer prodrug that can serve as a model compound for further development. We believe these novel aromatic nitrogen mustards will inspire further and effective applications.

The Leaving Group Strongly Affects H2O2-Induced DNA Cross-Linking by Arylboronates

We evaluated the effects of the benzylic leaving group and core structure of arylboronates on H2O2-induced formation of bisquinone methides for DNA interstrand cross-linking. The mechanism of DNA cross-linking induced by these arylboronates involves generation of phenol intermediates followed by departure of benzylic leaving groups leading to QMs which directly cross-link DNA via alkylation. The QM formation is the rate-determining step for DNA cross-linking. A better leaving group (Br) and stepwise bisquinone methide formation increased interstrand cross-linking efficiency. These findings provide essential guidelines for designing novel anticancer prodrugs.

Quantitative DNA interstrand cross-link formation by coumarin and thymine: structure determination, sequence effect, and fluorescence detection.

The coumarin analogues have been widely utilized in medicine, biology, biochemistry, and material sciences. Here, we report a detailed study on the reactivity of coumarins toward DNA. A series of coumarin analogues were synthesized and incorporated into oligodeoxynucleotides. A photoinduced [2 + 2] cycloaddition occurs between the coumarin moiety and the thymidine upon 350 nm irradiation forming both syn- and anti-cyclobutane adducts (17 and 18), which are photoreversible by 254/350 nm irradiation in DNA. Quantitative DNA interstrand cross-link (ICL) formation was observed with the coumarin moieties containing a flexible two-carbon or longer chain. DNA cross-linking by coumarins shows a kinetic preference when flanked by an A:T base pair as opposed to a G:C pair. An efficient photoinduced electron transfer between coumarin and dG slows down ICL formation. ICL formation quenches the fluorescence of coumarin, which, for the first time, enables fast, easy, and real-time monitoring of DNA cross-linking and photoreversibility via fluorescence spectroscopy. It can be used to detect the transversion mutation between pyrimidines and purines. Overall, this work provides new insights into the biochemical properties and possible toxicity of coumarins. A quantitative, fluorescence-detectable, and photoswitchable DNA cross-linking reaction of the coumarin moieties can potentially serve as mechanistic probes and tools for bioresearch without disrupting native biological environment.

UV-Induced DNA Interstrand Cross-Linking and Direct Strand Breaks from a New Type of Binitroimidazole Analogue.

Four novel photoactivated binitroimidazole prodrugs were synthesized. These agents produced DNA interstrand cross-links (ICLs) and direct strand breaks (DSB) upon UV irradiation, whereas no or very few DNA ICLs and DSBs were observed without UV treatment. Although these four molecules (1-4) contain the same binitroimidazole moiety, they bear four different leaving groups, which resulted in their producing different yields of DNA damage. Compound 4, with nitrogen mustard as a leaving group, showed the highest ICL yield. Surprisingly, compounds 1-3, without any alkylating functional group, also induced DNA ICL formation, although they did so with lower yields, which suggested that the binitroimidazole moiety released from UV irradiation of 1-3 is capable of cross-linking DNA. The DNA cross-linked products induced by these compounds were completely destroyed upon 1.0 M piperidine treatment at 90 °C (leading to cleavage at dG sites), which revealed that DNA cross-linking mainly occurred via alkylation of dGs. We proposed a possible mechanism by which alkylating agents were released from these compounds. HRMS and NMR analysis confirmed that free nitrogen mustards were generated by UV irradiation of 4. Suppression of DNA ICL and DSB formation by a radical trap, TEMPO, indicated the involvement of free radicals in the photo reactions of 3 and 4 with DNA. On the basis of these data, we propose that UV irradiation of compounds 1-4 generated a binitroimidazole intermediate that cross-links DNA. The higher ICL yield observed with 4 resulted from the amine effector nitrogen mustard released from UV irradiation.