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Nature Communications Jul 2018Cyclopropanation of unactivated olefinic bonds via addition of a reactive one-carbon species is well developed in synthetic chemistry, whereas natural cyclopropane...
Cyclopropanation of unactivated olefinic bonds via addition of a reactive one-carbon species is well developed in synthetic chemistry, whereas natural cyclopropane biosynthesis employing this strategy is very limited. Here, we identify a two-component cyclopropanase system, composed of a HemN-like radical S-adenosyl-L-methionine (SAM) enzyme C10P and a methyltransferase C10Q, catalyzes chemically challenging cyclopropanation in the antitumor antibiotic CC-1065 biosynthesis. C10P uses its [4Fe-4S] cluster for reductive cleavage of the first SAM to yield a highly reactive 5'-deoxyadenosyl radical, which abstracts a hydrogen from the second SAM to produce a SAM methylene radical that adds to an sp-hybridized carbon of substrate to form a SAM-substrate adduct. C10Q converts this adduct to CC-1065 via an intramolecular S2 cyclization mechanism with elimination of S-adenosylhomocysteine. This cyclopropanation strategy not only expands the enzymatic reactions catalyzed by the radical SAM enzymes and methyltransferases, but also sheds light on previously unnoticed aspects of the versatile SAM-based biochemistry.
Topics: Antibiotics, Antineoplastic; Bacterial Proteins; Biocatalysis; Cloning, Molecular; Cyclopropanes; Duocarmycins; Escherichia coli; Gene Expression; Genetic Vectors; Indoles; Iron; Iron-Sulfur Proteins; Methyltransferases; Recombinant Proteins; S-Adenosylhomocysteine; S-Adenosylmethionine; Streptomyces; Sulfur
PubMed: 30018376
DOI: 10.1038/s41467-018-05217-1 -
Experimental & Molecular Medicine May 2018Antibody-drug conjugates (ADCs) can selectively deliver cytotoxic agents to tumor cells and are frequently more potent than naked antibodies. However, optimization of...
Antibody-drug conjugates (ADCs) can selectively deliver cytotoxic agents to tumor cells and are frequently more potent than naked antibodies. However, optimization of the conjugation process between antibodies and cytotoxic agents and characterization of ADCs are laborious and time-consuming processes. Here, we describe a novel ADC platform using a tetravalent bispecific antibody that simultaneously binds to the tumor-associated antigen and a hapten conjugated to a cytotoxic agent. We selected cotinine as the hapten because it is not present in biological systems and is inert and nontoxic. We prepared an anti-epidermal growth factor receptor (EGFR) × cotinine bispecific antibody and mixed it with an equimolar amount of cotinine-conjugated duocarmycin to form the ADC. This ADC showed significant in vitro and in vivo antitumor activity against EGFR-positive, cetuximab-refractory lung adenocarcinoma cells with KRAS mutations.
Topics: Animals; Antibodies, Bispecific; Cell Death; Cell Line, Tumor; Cell Proliferation; Cetuximab; Cotinine; Disease Models, Animal; ErbB Receptors; Female; Humans; Indoles; Male; Mice, Inbred BALB C; Mutation; Proto-Oncogene Proteins p21(ras); Single-Chain Antibodies; Tissue Distribution; Xenograft Model Antitumor Assays
PubMed: 29795377
DOI: 10.1038/s12276-018-0096-z -
Acta Biochimica Et Biophysica Sinica May 2018
Topics: Antibiotics, Antineoplastic; Bacterial Proteins; Biological Products; Biosynthetic Pathways; Computational Biology; Drug Screening Assays, Antitumor; Duocarmycins; Genes, Bacterial; Indoles; Molecular Structure; Streptomyces
PubMed: 29659660
DOI: 10.1093/abbs/gmy030 -
Bioorganic & Medicinal Chemistry Letters Sep 2018New treatment modalities for glioblastoma multiforme (GBM) are urgently needed. Proton therapy is considered one of the most effective forms of radiation therapy for...
New treatment modalities for glioblastoma multiforme (GBM) are urgently needed. Proton therapy is considered one of the most effective forms of radiation therapy for GBM. DNA alkylating agents such as temozolomide (TMZ) are known to increase the radiosensitivity of GBM to photon radiation. TMZ is a fairly impotent agent, while duocarmycin SA (DSA) is an extremely potent cytotoxic agent capable of inducing a sequence-selective alkylation of duplex DNA. Here, the effects of sub-nM concentrations of DSA on the radiosensitivity of a human GBM cell line (U-138) to proton irradiation were examined. Radiation sensitivity was determined by viability, apoptosis, necrosis and clonogenic assays. DSA concentrations as low as 0.001 nM significantly sensitized U-138 cells to proton irradiation. DSA demonstrates synergistic cytotoxicity against GBM cells treated with proton radiation in vitro, which may represent a novel therapeutic alternative for the treatment of GBM.
Topics: Antibiotics, Antineoplastic; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Duocarmycins; Glioblastoma; Humans; Indoles; Necrosis; Protons; Pyrroles; Radiation-Sensitizing Agents
PubMed: 29650288
DOI: 10.1016/j.bmcl.2018.04.008 -
Oncotarget Jan 2018Small cell lung cancer (SCLC) is of a highly invasive and metastatic lung cancer subtype and there had not been effective targeted therapies. CD56, a cell surface marker...
Small cell lung cancer (SCLC) is of a highly invasive and metastatic lung cancer subtype and there had not been effective targeted therapies. CD56, a cell surface marker highly expressed on most SCLC, is a promising therapeutic target for treatment of this aggressive cancer. In this study, we generated a novel anti-CD56 antibody named promiximab, characterized by high affinity, internalization and tumor specificity. Then, the promiximab was conjugated with a potent DNA alkylating agent duocarmycin via reduced interchain disulfides to yield the promiximab-Duocarmycin (promiximab-DUBA) conjugates. Mass spectrometry analysis showed promiximab-DUBA had an average DAR (Drug-to-Antibody Ratio) of about 2.04. , promiximab-DUBA exerted strong inhibitory effects on SCLC cell lines NCI-H526, NCI-H524 and NCI-H69, with IC50 values of 0.07 nmol/L, 0.18 nmol/L and 0.29 nmol/L, respectively. antitumor activity, promiximab-DUBA at the dose of 5 mg/kg and 10 mg/kg every three days with a total of three times were sufficient to induce sustained regression of NCI-H526 tumors over control treatment with promiximab. Mostly, no recurrence was observed until 65 days post treatment with promiximab-DUBA. In the NCI-H69 subcutaneous xenograft model, significant inhibition of tumor growth was also observed following administration of promiximab-DUBA at the dose of 5 mg/kg or 10 mg/kg. Moreover, body weight and histopathology of major organs (liver, spleen, heart, lung and kidney) showed no significant changes after treatment of promiximab-DUBA. In conclusion, promiximab-DUBA is highly efficacious in small cell lung cancer xenograft models, and provides a new immunotherapy approach for SCLC.
PubMed: 29435172
DOI: 10.18632/oncotarget.23708 -
Chembiochem : a European Journal of... Apr 2018Capillary electrophoresis, coupled with DNA 5' Texas Red labeling, was used to investigate the ability of MNase, Fe peplomycin, and duocarmycin B to access the...
Capillary electrophoresis, coupled with DNA 5' Texas Red labeling, was used to investigate the ability of MNase, Fe peplomycin, and duocarmycin B to access the nucleosome. Distinct accessibility patterns of these species to the nucleosome were observed. MNase was completely prevented from approaching the nucleosome core and exhibited a higher site specificity for targeting DNA sites located close to the core region. Intercalation of peplomycin in the nucleosomal core region was highly suppressed, but reaction sites located at the ends of the nucleosomal core remained accessible, which implied flexibility of the core DNA end. Duocarmycin B was able to enter and react in the core region, although its alkylating efficiency decreased significantly.
Topics: DNA; DNA Cleavage; Duocarmycins; Electrophoresis, Capillary; Ferrous Compounds; Indoles; Micrococcal Nuclease; Nucleosomes; Peplomycin; Pyrrolidinones
PubMed: 29334166
DOI: 10.1002/cbic.201700643 -
Molecular Cancer Therapeutics Mar 2018Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that uses an antibody-photoabsorber conjugate (APC). However, the effect of NIR-PIT can...
Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that uses an antibody-photoabsorber conjugate (APC). However, the effect of NIR-PIT can be enhanced when combined with other therapies. NIR photocaging groups, based on the heptamethine cyanine scaffold, have been developed to release bioactive molecules near targets after exposure to light. Here, we investigated the combination of NIR-PIT using panitumumab-IR700 (pan-IR700) and the NIR-releasing compound, CyEt-panitumumab-duocarmycin (CyEt-Pan-Duo). Both pan-IR700 and CyEt-Pan-Duo showed specific binding to the EGFR-expressing MDAMB468 cell line In studies, additional injection of CyEt-Pan-Duo immediately after NIR light exposure resulted in high tumor accumulation and high tumor-background ratio. To evaluate the effects of combination therapy , tumor-bearing mice were separated into 4 groups: (i) control, (ii NIR-PIT, (iii) NIR-release, (iv) combination of NIR-PIT and NIR-release. Tumor growth was significantly inhibited in all treatment groups compared with the control group ( < 0.05), and significantly prolonged survival was achieved ( < 0.05 vs. control). The greatest therapeutic effect was shown with NIR-PIT and NIR-release combination therapy. In conclusion, combination therapy of NIR-PIT and NIR-release enhanced the therapeutic effects compared with either NIR-PIT or NIR-release therapy alone. .
Topics: Alkylating Agents; Animals; Breast Neoplasms; Cell Line, Tumor; Combined Modality Therapy; Duocarmycins; Female; Humans; Immunoconjugates; Indoles; Infrared Rays; Mice, Nude; Molecular Targeted Therapy; Phototherapy; Pyrrolidinones; Tumor Burden; Xenograft Model Antitumor Assays
PubMed: 29237807
DOI: 10.1158/1535-7163.MCT-17-0851 -
Journal of Nuclear Medicine : Official... Mar 2018Prostate cancer (PCa) is the most common cancer in men worldwide. In general, PCa responds poorly to chemotherapy. Therefore, antibody-drug conjugates (ADCs) have been...
Characterization of Site-Specifically Conjugated Monomethyl Auristatin E- and Duocarmycin-Based Anti-PSMA Antibody-Drug Conjugates for Treatment of PSMA-Expressing Tumors.
Prostate cancer (PCa) is the most common cancer in men worldwide. In general, PCa responds poorly to chemotherapy. Therefore, antibody-drug conjugates (ADCs) have been developed to specifically deliver highly cytotoxic drugs to the tumor. Because the prostate-specific membrane antigen (PSMA) is overexpressed in PCa, it represents a promising target for ADC-based therapies. The aim of this study was to evaluate the therapeutic efficacy of site-specifically conjugated duocarmycin- and monomethyl auristatin E (MMAE)-based anti-PSMA ADCs with drug-to-antibody ratios (DARs) of 2 and 4. The glycan group of the anti-PSMA antibody D2B was chemoenzymatically conjugated with duocarmycin or MMAE. Preservation of the immunoreactivity of the antibody on site-specific conjugation was investigated in vitro. Biodistribution and small-animal SPECT/CT imaging (18.5 ± 2.6 MBq) with 25 μg of In-labeled ADCs were performed on BALB/c nude mice with subcutaneous PSMA-positive LS174T-PSMA xenografts. Finally, the therapeutic efficacy of the 4 different ADCs was assessed in mice with LS174T-PSMA tumors. The immunoreactivity of the anti-PSMA antibody was preserved on site-specific conjugation. Biodistribution revealed high tumor uptake of all agents. The highest tumor uptake was observed in mice administered with In-D2B-DAR2-MMAE, reaching 119.7 ± 37.4 percentage injected dose per gram at 3 d after injection. Tumors of mice injected with In-D2B, In-D2B-DAR2-duocarmycin, In-D2B-DAR4-duocarmycin, In-D2B-DAR2-MMAE, and In-D2B-DAR4-MMAE could clearly be visualized with small-animal SPECT/CT. In contrast to unconjugated D2B or vehicle, treatment with either of the MMAE-based ADCs, but not with a duocarmycin-based ADC, significantly impaired tumor growth and prolonged median survival from 13 d (phosphate-buffered saline) to 20 and 29 d for DAR2 and DAR4 ADC, respectively. Tumor-doubling time increased from 3.5 ± 0.5 d to 5.2 ± 1.8 and 9.2 ± 2.1 d after treatment with D2B-DAR2-MMAE and D2B-DAR4-MMAE, respectively. The site-specifically conjugated anti-PSMA ADCs D2B-DAR2-MMAE and D2B-DAR4-MMAE efficiently targeted PSMA-expressing xenografts, effectively inhibited tumor growth of PSMA-expressing tumors, and significantly prolonged survival of mice.
Topics: Animals; Cell Line, Tumor; Cell Transformation, Neoplastic; Duocarmycins; Gene Expression Regulation, Neoplastic; Glutamate Carboxypeptidase II; Immunoconjugates; Indoles; Male; Mice; Mice, Nude; Oligopeptides; Prostatic Neoplasms; Pyrrolidinones; Single Photon Emission Computed Tomography Computed Tomography; Tissue Distribution
PubMed: 29146698
DOI: 10.2967/jnumed.117.196279 -
Nature Communications Nov 2017GyrI-like proteins are widely distributed in prokaryotes and eukaryotes, and recognized as small-molecule binding proteins. Here, we identify a subfamily of these...
GyrI-like proteins are widely distributed in prokaryotes and eukaryotes, and recognized as small-molecule binding proteins. Here, we identify a subfamily of these proteins as cyclopropanoid cyclopropyl hydrolases (CCHs) that can catalyze the hydrolysis of the potent DNA-alkylating agents yatakemycin (YTM) and CC-1065. Co-crystallography and molecular dynamics simulation analyses reveal that these CCHs share a conserved aromatic cage for the hydrolytic activity. Subsequent cytotoxic assays confirm that CCHs are able to protect cells against YTM. Therefore, our findings suggest that the evolutionarily conserved GyrI-like proteins confer cellular protection against diverse xenobiotics via not only binding, but also catalysis.
Topics: Alkylating Agents; Animals; Bacterial Physiological Phenomena; Binding Sites; Biocatalysis; Cell Line, Tumor; Crystallography, X-Ray; DNA; DNA Topoisomerases, Type II; Drug Resistance, Bacterial; Duocarmycins; Escherichia coli; Escherichia coli Proteins; Humans; Hydrolases; Hydrolysis; Indoles; Inhibitory Concentration 50; Jurkat Cells; Mice; Microbial Sensitivity Tests; Molecular Dynamics Simulation; Pyrroles; Recombinant Proteins; Repressor Proteins; Sequence Homology, Amino Acid; Streptomyces; Xenobiotics
PubMed: 29133784
DOI: 10.1038/s41467-017-01508-1 -
Oncotarget Sep 2017The endosialin/CD248/TEM1 receptor is expressed on the cell surface of tumor-associated stroma cells as well as in sarcoma and neuroblastoma cells. This receptor is...
The endosialin/CD248/TEM1 receptor is expressed on the cell surface of tumor-associated stroma cells as well as in sarcoma and neuroblastoma cells. This receptor is emerging as an attractive molecule in diagnostics and therapeutics because of its expression across the stroma of many human tumors, the low to absent expression in normal tissues and accessibility from the vascular circulation. In this study, we present evidence of the preclinical efficacy of a novel Antibody-Drug Conjugate (ENDOS/ADC). It consists of a humanized endosialin monoclonal antibody, named hMP-E-8.3, conjugated to a potent duocarmycin derivative. In endosialin expressing cancer cell lines, this ENDOS/ADC showed a powerful, specific and target-dependent killing activity. High expression levels of endosialin in cells correlated with efficient internalization and cytotoxic effects . Efficacy studies demonstrated that ENDOS/ADC treatment led to a long-lasting tumor growth inhibition of a cell line-based model of human osteosarcoma. Taken together, our results demonstrate that endosialin is an attractive target in sarcoma and suggest that ENDOS/ADC has the potential to be developed into a bio-therapeutic agent for these malignancies.
PubMed: 28947977
DOI: 10.18632/oncotarget.19499