Discovery of Cell‐Permeable Inhibitors That Target the BRCT Domain of BRCA1 Protein by Using a Small‐Molecule Microarray

BRCTs are phosphoserine‐binding domains found in proteins involved in DNA repair, DNA damage response and cell cycle regulation. BRCA1 is a BRCT domain‐containing, tumor‐suppressing protein expressed in the cells of breast and other human tissues. Mutations in BRCA1 have been found in ca. 50 % of hereditary breast cancers. Cell‐permeable, small‐molecule BRCA1 inhibitors are promising anticancer agents, but are not available currently. Herein, with the assist of microarray‐based platforms, we have discovered the first cell‐permeable protein–protein interaction (PPI) inhibitors against BRCA1. By targeting the (BRCT)₂ domain, we showed compound 15 a and its prodrug 15 b inhibited BRCA1 activities in tumor cells, sensitized these cells to ionizing radiation‐induced apoptosis, and showed synergistic inhibitory effect when used in combination with Olaparib (a small‐molecule inhibitor of poly‐ADP‐ribose polymerase) and Etoposide (a small‐molecule inhibitor of topoisomerase II). Unlike previously reported peptide‐based PPI inhibitors of BRCA1, our compounds are small‐molecule‐like and could be directly administered to tumor cells, thus making them useful for future studies of BRCA1/PARP‐related pathways in DNA damage and repair response, and in cancer therapy.     

DNA repair inhibition and cancer therapy.

The DNA repair process in mammalian cells is a multi-pathway mechanism that protects cells from the plethora of DNA damaging agents that are known to attack nuclear DNA. Moreover, the majority of current anticancer therapies (e.g. ionising radiation and chemotoxic therapies) rely on this ability to create DNA lesions, leading to apoptosis/cell death. A cells natural ability to repair such DNA damage is a major cause of resistance to these existing antitumour agents. It seems logical, therefore, that by modulating these repair mechanisms, greater killing effect to anticancer agents would occur. Experimental data support this, either through knockout studies or by the use of pharmacological inhibitors which target some of the key regulatory proteins involved in the DNA repair process. Several of these key DNA repair proteins which are actively under investigation as novel sites for intervention in cancer biology are discussed.     

Alternate therapeutic pathways for PARP inhibitors and potential mechanisms of resistance

Homologous recombination (HR) repair deficiency impairs the proper maintenance of genomic stability, thus rendering cancer cells vulnerable to loss or inhibition of DNA repair proteins, such as poly(ADP-ribose) polymerase-1 (PARP-1). Inhibitors of nuclear PARPs are effective therapeutics for a number of different types of cancers. Here we review key concepts and current progress on the therapeutic use of PARP inhibitors (PARPi). PARPi selectively induce synthetic lethality in cancer cells with homologous recombination deficiencies (HRDs), the most notable being cancer cells harboring mutations in the BRCA1 and BRCA2 genes. Recent clinical evidence, however, shows that PARPi can be effective as cancer therapeutics regardless of BRCA1/2 or HRD status, suggesting that a broader population of patients might benefit from PARPi therapy. Currently, four PARPi have been approved by the Food and Drug Administration (FDA) for the treatment of advanced ovarian and breast cancer with deleterious BRCA mutations. Although PARPi have been shown to improve progression-free survival, cancer cells inevitably develop resistance, which poses a significant obstacle to the prolonged use of PARP inhibitors. For example, somatic BRCA1/2 reversion mutations are often identified in patients with BRCA1/2-mutated cancers after treatment with platinum-based therapy, causing restoration of HR capacity and thus conferring PARPi resistance. Accordingly, PARPi have been studied in combination with other targeted therapies to overcome PARPi resistance, enhance PARPi efficacy, and sensitize tumors to PARP inhibition. Moreover, multiple clinical trials are now actively underway to evaluate novel combinations of PARPi with other anticancer therapies for the treatment of PARPi-resistant cancer. In this review, we highlight the mechanisms of action of PARP inhibitors with or without BRCA1/2 defects and provide an overview of the ongoing clinical trials of PARPi. We also review the current progress on PARPi-based combination strategies and PARP inhibitor resistance.Subject terms: Cancer therapy, Mechanisms of disease, PolyADP-ribosylation     

A bicyclic monoterpene diol and UVB stimulate BRCA1 phosphorylation in human keratinocytes

BRCA1 (breast cancer-associated gene 1) is a tumor suppressor gene that plays a role in DNA repair when phosphorylated. Many DNA-damaging agents including UVC and hydrogen peroxide have been shown to induce phosphorylation of BRCA1. Results of this study now show that both UVB and a bicyclic monoterpene diol (BMT diol) result in phosphorylation of BRCA1. This phosphorylation was maximal 2 h after treatment with either agent and declined to basal levels by 24 h. Inhibitor studies revealed that both UVB and the BMT diol phosphorylate BRCA1 through the FK506-binding protein-FKBP rapamycin-associated binding protein pathway, but the BMT diol also led to phosphorylation of BRCA1 through casein kinase II. This suggests that the signaling pathways for UVB and the BMT diol may diverge. Results of this study also show that the BMT diol stimulates the repair of UVB-induced cyclobutane pyrimidine dimers (CPD). Inhibitors of BMT diol-induced BRCA1 phosphorylation blocked the BMT diol-stimulated repair of CPD. This indicates that the BMT diol induces the phosphorylation of BRCA1, which, in turn, leads to an increase in repair of UVB-induced CPD. Therefore, this BMT diol may be useful for ameliorating the damaging effects of UVB.     

Planispine A Sensitized Cancer Cells to Cisplatin by Inhibiting the Fanconi Anemia Pathway

The use of cisplatin as a chemotherapeutic drug is impeded by the development of drug resistance. Combination therapies of a chemosensitizer for cisplatin have been studied, but with little success, and the search for an effective combination therapy is continuing. Our earlier reports have shown that Zanthoxylum armatum DC. extract enhances the apoptotic effect of cisplatin in cancer cell lines. In this study, we purified and identified the bioactive phytocompound through bio-assay-guided purification, using column chromatography and HPLC. Chemical characterization using NMR and mass spectrometry revealed the compound as planispine A, with molecular structure C₂₅H₃₀O₆ and molecular weight, 426.16 g/mol. Planispine A was found to inhibit cancer cell proliferation in a dose-dependent manner and to sensitize the cancer cells to cisplatin-augmented apoptotic cell death, in a caspase-dependent manner. A combination of planispine A and cisplatin induced S-phase cell cycle arrest, and reduced the expression of survival proteins such as cyclin D1. Interestingly, planispine A inhibits the Fanconi anemia pathway, as shown by reduced FANCD2 foci formation and FANCD2 monoubiquitination, which revealed the molecular mechanism of chemo-sensitization of cancer cells to cisplatin. Evaluation of this combination therapy in cisplatin-resistant tumors may lead to more efficient cisplatin treatment.     

A photoreactive small-molecule probe for 2-oxoglutarate oxygenases

2-oxoglutarate (2-OG)-dependent oxygenases have diverse roles in human biology. The inhibition of several 2-OG oxygenases is being targeted for therapeutic intervention, including for cancer, anemia, and ischemic diseases. We report a small-molecule probe for 2-OG oxygenases that employs a hydroxyquinoline template coupled to a photoactivable crosslinking group and an affinity-purification tag. Following studies with recombinant proteins, the probe was shown to crosslink to 2-OG oxygenases in human crude cell extracts, including to proteins at endogenous levels. This approach is useful for inhibitor profiling, as demonstrated by crosslinking to the histone demethylase FBXL11 (KDM2A) in HEK293T nuclear extracts. The results also suggest that small-molecule probes may be suitable for substrate identification studies.     

Rapid mutation detection in complex genes by heteroduplex analysis with capillary array electrophoresis

Mutational analysis of large multiexon genes without prevalent mutations is a laborious undertaking that requires the use of a high-throughput scanning technique. The Human Genome Project has enabled the development of powerful techniques for mutation detection in large multiexon genes. We have transferred heteroduplex analysis (HA) by conformation-sensitive gel electrophoresis of the two major breast cancer (BC) predisposing genes, BRCA1 and BRCA2, to a multicapillary DNA sequencer in order to increase the throughput of this technique. This new method that we have called heteroduplex analysis by capillary array electrophoresis (HA-CAE) is based on the use of multiplex-polymerase chain reaction (PCR), different fluorescent labels and HA in a 16-capillary DNA sequencer. To date, a total of 114 different DNA sequence variants (19 insertions/deletions and 95 single-nucleotide substitutions - SNS) of BRCA1 and BRCA2 from 431 unrelated BC families have been successfully detected by HA-CAE. In addition, we have optimized the multiplex-PCR conditions for the colorectal cancer genes MLH1 and MSH2 in order to analyze them by HA-CAE. Both genes have been amplified in 13 multiplex groups, which contain the 35 exons, and their corresponding flanking intronic sequences. MLH1 and MSH2 have been analyzed in nine hereditary nonpolyposis colorectal cancer patients, and we have found six different DNA changes: one complex deletion/insertion mutation in MLH1 exon 19 and another five SNS. Only the complex mutation and one SNS may be classified as cancer-prone mutations. Our experience has revealed that HA-CAE is a simple, fast, reproducible and sensitive method to scan the sequences of complex genes.     

Mitochondrial alterations in fanconi anemia fibroblasts following ultraviolet A or psoralen photoactivation

The genetic disease Fanconi anemia (FA), generally considered to be a DNA repair defect, has also been related to a deficiency in cellular defense against reactive oxygen species (ROS). Results show that mitochondrial matrix densification occurs rapidly and transiently in FA fibroblasts following 8-methoxypsoralen (8-MOP) photoreaction or ultraviolet A (320 to 380 nm) (UVA) irradiation. This effect is oxygen dependent because it is more important under 20 than under 5% oxygen tension. In contrast, in normal fibroblasts very little, if any, densification of mitochondrial matrix is induced by treatments even at the highest oxygen tension. The changes in matrix density in FA cells are accompanied by some modifications in transmembrane potential, linked to a Fenton-like reaction, and in mitochondrial cardiolipin content, differing from the responses of normal cells. These data are indicative of some sort of membrane damage induced by 8-MOP photoreaction and UVA irradiation, to which FA cells appear to be particularly sensitive.     

泛素-蛋白酶体与药物应用

In eukaryotes, the ubiquitin-proteasome pathway degrades the majority of intracellular proteins tagged with polyubiquitin chains. It participates in regulation of key cellular activities, such as cell proliferation, cell differentiation, apoptosis, DNA repair, etc. through the degradation of malformed or misfolded proteins. Dysfunctions of the ubiquitin-proteasome pathway have been linked to many diseases, including cancer and neurodegeneration, etc. The commercially available proteasome inhibitors have been successfully used to treat multiple myeloma and mantle cell lymphoma. In addition, novel inhibitors against other components of the ubiquitin-proteasome pathway, such as those enzymes that drive ubiquitination and deubiquitination in preclinical testing or clinical trials, exhibit promising therapeutic effects in vivo. This paper briefly introduces the ubiquitin-proteasome pathway related drug discovery progress.     

A microarray to measure repair of damaged plasmids by cell lysates

DNA repair mechanisms constitute major defences against agents that cause cancer, degenerative disease and aging. Different repair systems cooperate to maintain the integrity of genetic information. Investigations of DNA repair involvement in human pathology require an efficient tool that takes into account the variety and complexity of repair systems. We have developed a highly sensitive damaged plasmid microarray to quantify cell lysate excision/synthesis (ES) capacities using small amounts of proteins. This microsystem is based on efficient immobilization and conservation on hydrogel coated glass slides of plasmid DNA damaged with a panel of genotoxic agents. Fluorescent signals are generated from incorporation of labelled dNTPs by DNA excision-repair synthesis mechanisms at plasmid sites. Highly precise DNA repair phenotypes i.e. simultaneous quantitative measures of ES capacities toward seven lesions repaired by distinct repair pathways, are obtained. Applied to the characterization of xeroderma pigmentosum (XP) cells at basal level and in response to a low dose of UVB irradiation, the assay showed the multifunctional role of different XP proteins in cell protection against all types of damage. On the other hand, measurement of the ES of peripheral blood mononuclear cells from six donors revealed significant diversity between individuals. Our results illustrate the power of such a parallelized approach with high potential for several applications including the discovery of new cancer biomarkers and the screening of chemical agents modulating DNA repair systems.     

FR900482 class of anti-tumor drugs cross-links oncoprotein HMG I/Y to DNA in vivo

BACKGROUND: Overexpression of the high-mobility group, HMG I/Y, family of chromatin oncoproteins has been implicated as a clinical diagnostic marker for both neoplastic cellular transformation and increased metastatic potential of several human cancers. These minor groove DNA-binding oncoproteins are thus an attractive target for anti-tumor chemotherapy. FR900482 represents a new class of anti-tumor agents that bind to the minor groove of DNA and exhibit greatly reduced host toxicity compared to the structurally related mitomycin C class of anti-tumor drugs. We report covalent cross-linking of DNA to HMG I/Y by FR900482 in vivo which represents the first example of a covalent DNA-drug-protein cross-link with a minor groove-binding oncoprotein and a potential novel mechanism through which these compounds exert their anti-tumor activity. RESULTS: Using a modified chromatin immunoprecipitation procedure, fragments of DNA that have been covalently cross-linked by FR900482 to HMG I/Y proteins in vivo were polymerase chain reaction-amplified, isolated and characterized. The nuclear samples from control cells were devoid of DNA fragments whereas the nuclear samples from cells treated with FR900482 contained DNA fragments which were cross-linked by the drug to the minor groove-binding HMG I/Y proteins in vivo. Additional control experiments established that the drug also cross-linked other non-oncogenic minor groove-binding proteins (HMG-1 and HMG-2) but did not cross-link major groove-binding proteins (Elf-1 and NFkappaB) in vivo. Our results are the first demonstration that FR900482 cross-links a number of minor groove-binding proteins in vivo and suggests that the cross-linking of the HMG I/Y oncoproteins may participate in the mode of efficacy as a chemotherapeutic agent. CONCLUSIONS: We have illustrated that the FR class of anti-tumor antibiotics, represented in this study by FR900482, is able to produce covalent cross-links between the HMG I/Y oncoproteins and DNA in vivo. The ability of this class of compounds to cross-link the HMG I/Y proteins in the minor groove of DNA represents the first demonstration of drug-induced cross-linking of a specific cancer-related protein to DNA in living cells. We have also demonstrated that FR900482 cross-links other minor groove-binding proteins (HMG-1 and HMG-2 in the present study) in vivo; however, since HMG I/Y is the only minor groove-binding oncoprotein presently known, it is possible that these non-histone chromatin proteins are among the important in vivo targets of this family of drugs. These compounds have already been assessed as representing a compelling clinical replacement for mitomycin C due to their greatly reduced host toxicity and superior DNA interstrand cross-linking efficacy. The capacity of FR900482 to cross-link the HMG I/Y oncoprotein with nuclear DNA in vivo potentially represents a significant elucidation of the anti-tumor efficacy of this family of anticancer agents.     

TOXICITY, DNA DAMAGE AND INHIBITION OF DNA REPAIR SYNTHESIS IN HUMAN MELANOMA CELLS BY CONCENTRATED SUNLIGHT

A 1 m diameter water lens was used to focus solar radiation, giving an 8-fold concentration of the total spectrum and a cytocidal flux similar to that of laboratory UV sources. Survival curves for human melanoma cells were similar for sunlight and 254 nm UV, in that D _q, was usually larger than D _o. An xeroderma pigmentosum lymphoblastoid line was equally sensitive to both agents and human cell lines sensitive to ionizing radiation (lymphoblastoid lines), crosslinking agents or monofunctional alkylating agents (melanoma lines) had the same 254 nm UV and solar survival responses as appropriate control lines. Two melanoma sublines derived separately by 16 cycles of treatment with sunlight or 254 nm UV were crossresistant to both agents. In one melanoma cell line used for further studies, DNA strand breaks and DNA-protein crosslinking were induced in melanoma cells by sunlight but pyrimidine dimers (paper chromatography) and DNA interstrand crosslinking (alkaline elution) could not be detected. The solar fiuence response of DNA repair synthesis was much less than that from equitoxic 254 nm UV, reaching a maximum near the D _o value and then declining; semiconservative DNA synthesis on the other hand remained high. These effects were not due to changes in thymidine pool sizes. Solar exposure did not have a major effect on 254 nm UV-induced repair synthesis.     

The role of dUTPase and uracil-DNA repair in cancer chemotherapy

Thymidylate metabolism is an important target for chemotherapeutic agents that combat a variety of neoplastic diseases including head and neck, breast and gastrointestinal cancers. Therapeutic strategies applied to this pathway target the thymidylate synthase (TS) reaction that catalyzes the reductive methylation of deoxyuridylate (dUMP) to form thymidylate (TMP). This reaction represents the sole de novo source of TMP required for DNA replication and repair. Inhibitors of this pathway include the widely utilized fluoropyrimide and antifolate classes of anti-cancer agents. Studies attempting to elucidate the molecular mechanisms of cell killing mediated by inhibitors of the TS reaction suggest that cytotoxicity results from a process known as "thymineless death". This term describes the extreme TTP pool depletion observed following TS inhibition. Although depletion of TTP pools is clearly involved in this process, there is now considerable evidence implicating aberrant uracil-DNA metabolism as an important mechanism of toxicity. Upon TS inhibition, dUTP pools may accumulate, inducing repeated cycles of uracil misincorporation into DNA and repair-mediated DNA damage. Central to the uracil-misincorporation pathway are the enzymes deoxyuridine nucleotidohydrolase (dUTPase) (EC 3.6.1.23) and uracil-DNA glycoslyase (UDG) (EC 3.2.2.3). dUTPase catalyzes the hydrolysis of dUTP to form dUMP and pyrophosphate thereby eliminating dUTP and preventing its utilization by DNA polymerases during replication and repair. UDG initiates the base excision repair pathway effectively removing any uracil residues that may arise in DNA. Under normal conditions, uracil is precluded from DNA by the combined actions of dUTPase and UDG. However, during TS inhibition, dUTP pools may accumulate and overwhelm dUTPase, resulting in repeated cycles of uracil misincorporation and detrimental repair leading to strand breaks and cell death. Because dUTPase plays a pivotal role in regulating cellular dUTP pools, this enzyme could have profound effects on the efficacy of agents that target thymidylate biosynthesis. This article reviews our current understanding of the role of aberrant uracil-DNA metabolism as a contributing mechanism of cytotoxicity initiated by chemotherapeutic agents that target de novo thymidylate metabolism. The role of dUTPase expression in modulating therapeutic response is presented including evidence from yeast and mammalian cell culture models and clinical studies. The regulation of human dUTPase isoforms in normal and neoplastic tissues will be reviewed as well as the role of dUTPase expression as a prognostic marker for overall survival and response to therapy in colon cancer.     

Computational and experimental studies of the interaction between phospho-peptides and the C-terminal domain of BRCA1

The C-terminal domain of BRCA1(BRCT) is involved in the DNA repair pathway by recognizing the pSXXF motif in interacting proteins. It has been reported that short peptides containing this motif bind to BRCA1(BRCT) in the micromolar range with high specificity. In this work, the binding of pSXXF peptides has been studied computationally and experimentally in order to characterize their interaction with BRCA1(BRCT). Elucidation of the contacts that drive the protein–ligand interaction is critical for the development of high affinity small-molecule BRCA1 inhibitors. Molecular dynamics (MD) simulations revealed the key role of threonine at the peptide P+2 position in providing structural rigidity to the ligand in the bound state. The mutation at P+1 had minor effects. Peptide extension at the N-terminal position with the naphthyl amino acid exhibited a modest increase in binding affinity, what could be explained by the dispersion interaction of the naphthyl side-chain with a hydrophobic patch. Three in silico end-point methods were considered for the calculation of binding free energy. The Molecular Mechanics Poisson–Boltzmann Surface Area and the Solvated Interaction Energy methods gave reasonable agreement with experimental data, exhibiting a Pearlman predictive index of 0.71 and 0.78, respectively. The MM-quantum mechanics-surface area method yielded improved results, which was characterized by a Pearlman index of 0.78. The correlation coefficients were 0.59, 0.61 and 0.69, respectively. The ability to apply a QM level of theory within an end-point binding free energy protocol may provide a way for a consistent improvement of accuracy in computer-aided drug design.