Bleomycins Research Articles

Pulmonary Delivery of Recombinant Human Bleomycin Hydrolase Using Mannose-Modified Hierarchically Porous UiO-66 for Preventing Bleomycin-Induced Pulmonary Fibrosis.

Bleomycins (BLMs) are widely used in clinics as antitumor agents. However, BLM-based chemotherapies often accompany severe pulmonary fibrosis (PF). Human bleomycin hydrolase is a cysteine protease that can convert BLMs into inactive deamido-BLMs. In this study, mannose-modified hierarchically porous UiO-66 (MHP-UiO-66) nanoparticles (NPs) were used to encapsulate the recombinant human bleomycin hydrolase (rhBLMH). When rhBLMH@MHP-UiO-66 was intratracheally instilled into the lungs, the NPs were transported into the epithelial cells, and rhBLMH prevented the lungs from PF during BLM-based chemotherapies. Encapsulation of rhBLMH in the MHP-UiO-66 NPs protects the enzyme from proteolysis in physiological conditions and enhances cellular uptake. In addition, the MHP-UiO-66 NPs significantly enhance the pulmonary accumulation of intratracheally instilled rhBLMH, thus providing more efficient protection of the lungs against BLMs during the chemotherapies.

The Structure-Function Relationship of Human Bleomycin Hydrolase: Mutation of a Cysteine Protease into a Serine Protease.

Human bleomycin hydrolase (hBH) catalyzes deamidation of the anticancer drug bleomycins (BLM). This enzyme is involved in BLM detoxification and drug resistance. Herein, we report the putative BLM-binding site and catalytic mechanism of hBH. The crystal structures and biochemical studies suggest that hBH cleaves its C-terminal residue without significant preference for the type of amino acid, and therefore can accordingly accommodate the β-aminoalanine amide moiety of BLM for deamidation. Interestingly, hBH is capable of switching from a cysteine protease to a serine protease that is unable to cleave the secondary amide of hBH C-terminus but reacts with the primary amide of BLMs.

Nanopore single-molecule detection of bleomycin via dumbbell DNA scission

It’s of great significance to develop convenient and sensitive quantitative detection assays for the bleomycins (BLMs) as the BLMs are widely used in clinical treatment. In the present work, the authors described a new method for BLM detection using dumbbell DNA as probe on the nanopore sensor platform, which is based on the scission function on DNA by BLM. A dumbbell DNA with TTTTT-five bases loop was chosen due to its longer dwell time and higher frequency of the blockages in nanopore. With addition of BLM·Fe(II), the blockage events of the dumbbell DNA gradually transfer to single strand DNA (ssDNA) fragment signals, and it turns out to be a quantitative relationship between event intervals (τon) and the concentration of BLM in a certain range, making it a promising method for quantitative detection of BLMs with high selectivity and ultra-low detection limit of 0.1 nM.

Deoxidized gulose moiety attenuates the pulmonary toxicity of 6'-deoxy-bleomycin Z without effect on its antitumor activity

Bleomycins (BLMs) are broad-spectrum antitumor drugs, but the dose-dependent lung toxicity has restricted their therapeutic applications. Many efforts have contributed to develop novel BLM analogues, but mainly focused on single functional domain owing to the structural complexity of BLM. Benefit from the engineered production of two novel analogues 6'-deoxy-BLM Z (6'-DO-BLM Z) and BLM Z, they together with clinical BLM-sulfate comprised a good model with varied sugar or C-terminal domain in any two of them, allowing us to study their structure-activity relationships pairwise. Our investigations suggested the biological activities of BLM or its analogues are mainly depended on the C-terminal amine, while the changed C-terminal amine endowed BLM Z with much higher pulmonary toxicity comparing to BLM-sulfate, whereas the deoxidized gulose unit with same C-terminal amine evidently attenuated the pulmonary toxicity of 6'-DO-BLM Z without effect on antitumor activity. Further mechanistic studies revealed that the alleviation of pulmonary toxicity in 6'-DO-BLM Z by a slight change in the sugar moiety could attribute to the decrease of ROS production and thereby reduce the subsequent caspase-1 activity and resulting inflammatory response. Therefore, the synergistic modifications on C-terminal amine and sugar moiety provide new insights to efficiently develop potential BLM candidate with good clinical performance.

Synthesis of an Alkyne‐Modified Bleomycin Disaccharide Precursor, Conversion to a 18F‐Labeled Radiotracer, and Preliminary in vivo‐PET Imaging Studies

The bleomycins (BLMs) are known antitumor antibiotics composed of the tumoricidal and tumor seeking domains. The peptide structure of BLMs is responsible for the cytotoxicity by selective oxidative cleavage of DNA (and RNA), while the tumor cell selectivity and internalization resides in the disaccharide moiety (i.e. BLM disaccharide). This has prompted researchers to utilize BLM disaccharide and its derivatives as constituents for the selective recognition of tumor cells, which may find further applications as new tumor imaging tools or drug delivery vehicles. In the present study a high yielding synthesis of an alkyne modified BLM disaccharide precursor that may be used as a useful agent for the click conjugation, its conversion to a 18F‐labeled radiotracer, and preliminary in vivo PET imaging studies of the tracer with breast cancer (MCF‐7) xenograft mouse models are described.

Importance of Metal-Ion Exchange for the Biological Activity of Coordination Complexes of the Biomimetic Ligand N4Py.

Metal coordination complexes can display interesting biological activity, as illustrated by the bleomycins (BLMs), a family of natural antibiotics that when coordinated to a redox-active metal ion, show antitumor activity. Yet, which metal ion is required for the activity in cells is still subject to debate. In this study, we described how different metal ions affect the intracellular behavior and activity of the synthetic BLM-mimic N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine (N4Py). Our study shows that a mixture of iron(II), copper(II), and zinc(II) complexes can be generated when N4Py is added to cell cultures but that the metal ion can also be exchanged by other metal ions present in cells. Moreover, the combination of chemical data, together with the performed biological experiments, shows that the active complex causing oxidative damage to cells is the FeII-N4Py complex and not per se the metal complex that was initially added to the cell culture medium. Finally, it is proposed that the high activity observed upon the addition of the free N4Py ligand is the result of a combination of scavenging of biologically relevant metals and oxidative damage caused by the iron(II) complex.

The discovery and development of microbial bleomycin analogues.

The bleomycins (BLMs) belong to a subfamily of glycopeptide antibiotics and are clinically applied in combination chemotherapy regimens to treat various malignancies. But the therapeutic applications of BLMs are restricted by the accompanied dose-dependent lung toxicity and potential incidence of lung fibrosis. Many efforts have been devoted to develop novel BLM analogues, for seeking of drug leads with improved antitumor activity and/or reduced lung toxicity. The progresses in the biosynthetic studies of BLMs have greatly expedited the process to achieve such goals. This review highlights the discovery and development of microbial BLM analogues in the past two decades, especially those derived from engineered biosynthesis. Moreover, the summarized structure-activity relationship, which is specifically focusing on the sugar moiety, shall shed new insights into the prospective development of BLM analogues.

Activities of recombinant human bleomycin hydrolase on bleomycins and engineered analogues revealing new opportunities to overcome bleomycin-induced pulmonary toxicity

The bleomycins (BLMs) are widely used in combination therapies for the treatment of various cancers. Dose-dependent and cumulative pulmonary toxicity is the major cause of BLM-associated morbidity, limiting the broad uses of BLMs as anticancer drugs. The organ specificity of BLM-induced toxicity has been correlated with the expression of the hBLMH gene, encoding the human bleomycin hydrolase (hBLMH), which is poorly expressed in the lung. hBLMH hydrolyzes BLMs into the biologically inactive deamido BLMs, thereby protecting organs from BLM-induced toxicity. Here we report (i) expression of hBLMH and production and isolation of recombinant human bleomycin hydrolase (rhBLMH) from E. coli, (ii) structural characterization of deamido BLM A2 and B2 isolated from rhBLMH-catalyzed hydrolysis of BLM A2 and B2, and (iii) kinetic characterization of the rhBLMH-catalyzed hydrolysis of BLM A2 and B2, in comparison with five BLM analogues. rhBLMH from E. coli catalyzes rapid and efficient hydrolysis of all BLMs tested, exhibiting a superior catalytic efficiency for BLM B2. These findings reveal new opportunities to overcome BLM-induced pulmonary toxicity in chemotherapies, potentially by exploring BLM B2 as the preferred congener, engineering designer BLMs with optimized activity for rhBLMH, or co-administrating rhBLMH directly into the lung as a potential protein therapeutic.

Strain improvement by combined UV mutagenesis and ribosome engineering and subsequent fermentation optimization for enhanced 6'-deoxy-bleomycin Z production.

The bleomycins (BLMs) are important clinical drugs extensively used in combination chemotherapy for the treatment of various cancers. Dose-dependent lung toxicity and the development of drug resistance have restricted their wide applications. 6'-Deoxy-BLM Z, a recently engineered BLM analogue with improved antitumor activity, has the potential to be developed into the next-generation BLM anticancer drug. However, its low titer in the recombinant strain Streptomyces flavoviridis SB9026 has hampered current efforts, which require sufficient compound, to pursue preclinical studies and subsequent clinical development. Here, we report the strain improvement by combined UV mutagenesis and ribosome engineering, as well as the fermentation optimization, for enhanced 6'-deoxy-BLM production. A high producer, named S. flavoviridis G-4F12, was successfully isolated, producing 6'-deoxy-BLM at above 70mg/L under the optimized fermentation conditions, representing a sevenfold increase in comparison with that of the original producer. These findings demonstrated the effectiveness of combined empirical breeding methods in strain improvement and set the stage for sustainable production of 6'-deoxy-BLM via pilot-scale microbial fermentation.

Engineered production and evaluation of 6'-deoxy-tallysomycin H-1 revealing new insights into the structure-activity relationship of the anticancer drug bleomycin.

The bleomycins (BLMs), a family of glycopeptide antibiotics, are currently used clinically in combination with a number of other agents for the treatment of malignant tumors. Other members of the BLM family include tallysomycins (TLMs), phleomycins and zorbamycin (ZBM). We previously cloned and characterized the biosynthetic gene clusters for BLMs, TLMs and ZBM. Applications of combinatorial biosynthesis strategies to the three biosynthetic machineries enabled the engineered production of several BLM analogs with unique structural characteristics and varying DNA cleavage activities, thereby providing an outstanding opportunity to study the structure-activity relationship (SAR) for the BLM family of anticancer drugs. We now report the engineered production of a new BLM-TLM-ZBM hybrid metabolite, named 6'-deoxy-TLM H-1, which consists of the 22-desmethyl-BLM aglycone, the TLM A C-terminal amine and the ZBM disaccharide, by heterologous expression of the zbmGL genes from the ZBM biosynthetic gene cluster in the Streptoalloteichus hindustanus ΔtlmH mutant strain SB8005. Evaluation of the DNA cleavage activities of 6'-deoxy-TLM H-1 as a measurement for its potential anticancer activity, in comparison with TLM H-1 and BLM A2, reveals new insight into the SAR of BLM family of anticancer drugs.The Journal of Antibiotics advance online publication, 23 August 2017; doi:10.1038/ja.2017.93.

Carbamoylmannose enhances the tumor targeting ability of supramolecular nanoparticles formed through host-guest complexation of a pair of homopolymers.

Conjugation of sugars to antitumor drugs can facilitate drug binding to tumor cells and the saccharide motifs of bleomycins (BLMs) play a crucial role in tumor-seeking. Here, we synthesized BLM monosaccharide, carbamoylmannose, and subsequently prepared carbamoylmannose decorated platinum-incorporating supramolecular nanoparticles formed through the host-guest complexation of poly(N-vinylpyrrolidone) and poly(aspartic acid). The targeting effects of carbamoylmannose decorated supramolecular nanoparticles in various cancer cells and tumor-bearing mice were investigated. It was found that the nanoparticles showed a specific in vitro and in vivo carbamoylmannose-mediated cellular uptake and drug delivery. The cellular uptake of the nanoparticles followed the receptor-mediated endocytosis mechanism in cancer cells but not in healthy cells. In a murine hepatic H22 tumor model, it was demonstrated that the carbamoylmannose moiety increased the plasma concentration, tumor targeting ability and tumor penetration of the nanoparticles, leading to high tumor accumulation and superior antitumor efficacy. This carbamoylmannose molecule may bring an opportunity to design and construct inexpensive but highly efficient drug and gene delivery systems in the future.

Targeted Delivery of Bleomycin: A Comprehensive Anticancer Review.

Despite being one of the most effective broad-spectrum chemotherapeutic agents in the treatment of cancers, the clinical applications of bleomycins (BLMs) have been limited due to their poor drug delivery abilities, and the side effect of causing lung fibrosis. With the increased therapeutics and the reduction of side effects, research and development of targeted drug delivery systems (TDDS) with BLMs have become essential for the expansive clinical usage of BLM-based therapeutics. This review summarizes the recent developments of various TDDS for BLMs, including techniques such as photochemical internalization, ultrasound, and micelle, liposome, and nanoparticle formation. The advantages and disadvantages for each delivery approach are outlined, along with the specific challenges associated with each delivery system.

Modified bleomycin disaccharides exhibiting improved tumor cell targeting.

The bleomycins (BLMs) are a family of antitumor antibiotics used clinically for anticancer chemotherapy. Their antitumor selectivity derives at least in part from their ability to target tumor cells, a property that resides in the carbohydrate moiety of the antitumor agent. In earlier studies, we have demonstrated that the tumor cell selectivity resides in the mannose carbamoyl moiety of the BLM saccharide and that both the BLM disaccharide and monosaccharide containing the carbamoyl moiety were capable of the delivery/uptake of a conjugated cyanine dye into cultured cancer cell lines. Presently, the nature of the participation of the carbamoyl moiety has been explored further to provide compounds of utility for defining the nature of the mechanism of tumor cell recognition and uptake by BLM saccharides and in the hope that more efficient compounds could be identified. A library of seven disaccharide–Cy5** dye conjugates was prepared that are structural analogues of the BLM disaccharide. These differed from the natural BLM disaccharide in the position, orientation, and substitution of the carbamoyl group. Studies of these compounds in four matched sets of tumor and normal cell lines revealed a few that were both tumor cell selective and internalized 2–4-fold more efficiently than the natural BLM disaccharide.

Electrochemical determination of bleomycins based on dual-amplification of 4-mercaptophenyl boronic acid-capped gold nanoparticles and dopamine-capped gold nanoparticles

Due to the significance of bleomycins (BLMs) in the treatment of a variety of cancers, it is highly desirable to develop a sensitive method to quantitatively determine the content of BLMs in both pharmaceutical analysis and clinical samples. Here, a simple, rapid, and convenient electrochemical DNA sensor for determination of BLMs was developed based on 4-mercaptophenyl boronic acid-capped gold nanoparticles (MBA-AuNPs) and dopamine-capped gold nanoparticles (DA-AuNPs) as a dual-amplification element. In the presence of Fe(II)·BLMs, the electrochemical DNA sensor undergoes an irreversible cleavage event, which can result in a significant decrease of the oxidation peak current of dopamine. This proposed sensor reveals a wide linear range from 0.07 nM to 910 nM (R = 0.9958) and a low detectable limit (0.02 nM) for the determination of BLMs. It also exhibits a good performance in serum samples. Based on the high sensitivity and specificity of this sensor, and the inherent characteristics (cost-effective and simple-to-implement) of the electrochemical technique, the developed DNA sensor shows distinct advantages over conventional methods and is a promising method for the determination of trace amounts of BLMs in both pharmaceutical analysis and clinical samples.

Efficient Nuclear DNA Cleavage in Human Cancer Cells by Synthetic Bleomycin Mimics

Iron complexes of N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)-methylamine (N4Py) have proven to be excellent synthetic mimics of the Bleomycins (BLMs), which are a family of natural antibiotics used clinically in the treatment of certain cancers. However, most investigations of DNA cleavage activity of these and related metal complexes were carried out in cell-free systems using plasmid DNA as substrate. The present study evaluated nuclear DNA cleavage activity and cell cytotoxicity of BLM and its synthetic mimics based on the ligand N4Py. The N4Py-based reagents induced nuclear DNA cleavage in living cells as efficiently as BLM and Fe(II)-BLM. Treatment of 2 cancer cell lines and 1 noncancerous cell line indicated improved cytotoxicity of N4Py when compared to BLM. Moreover, some level of selectivity was observed for N4Py on cancerous versus noncancerous cells. It was demonstrated that N4Py-based reagents and BLM induce cell death via different mechanistic pathways. BLM was shown to induce cell cycle arrest, ultimately resulting in mitotic catastrophe. In contrast, N4Py-based reagents were shown to induce apoptosis effectively. To the best of our knowledge, the present study is the first demonstration of efficient nuclear DNA cleavage activity of a synthetic BLM mimic within cells. The results presented here show that it is possible to design synthetic bioinorganic model complexes that are at least as active as the parent natural product and thereby are potentially interesting alternatives for BLM to induce antitumor activity.

Amplified impedimetric DNA sensor based on graphene oxide–phenylboronic acid for sensitive detection of bleomycins

Due to the significance of bleomycins (BLMs) for the treatment of a variety of cancers, it is highly desirable to develop a sensitive method to quantitatively determine the BLMs content in both pharmaceutical analysis and clinical samples. Here, a simple, rapid, and convenient electrochemical method for BLMs detection was developed. Single-stranded DNA, as the substrate of BLMs, was self-assembled onto a gold electrode to fabricate an electrochemical DNA sensor, with one terminus tethered on the electrode surface and the other terminus labeled with graphene oxide–phenylboronic acid as a signal-amplification factor, giving a remarkable electron-transfer resistance (Ret). In the presence of Fe(II)·BLMs, the electrochemical DNA sensor undergoes an irreversible cleavage event, which can be transduced into a significant decrease in Ret. This sensor reveals a wide linear range from 0.05 nM to 1200 nM (R = 0.9869) and a low detection limit (0.01 nM, S/N = 3) for the determination of BLMs. It exhibits a good performance in a serum sample as well. Based on the high sensitivity and specificity of this electrochemical DNA sensor and the attractive characteristics of the electrochemical technique (cost effective and simple to implement), the developed impedimetric DNA sensor shows distinct advantages over conventional methods and is a promising alternative for the sensitive determination of BLMs in clinical samples.

A Designer Bleomycin with Significantly Improved DNA Cleavage Activity

The bleomycins (BLMs) are used clinically in combination with a number of other agents for the treatment of several types of tumors, and the BLM, etoposide, and cisplatin treatment regimen cures 90-95% of metastatic testicular cancer patients. BLM-induced pneumonitis is the most feared, dose-limiting side effect of BLM in chemotherapy, which can progress into lung fibrosis and affect up to 46% of the total patient population. There have been continued efforts to develop new BLM analogues in the search for anticancer drugs with better clinical efficacy and lower lung toxicity. We have previously cloned and characterized the biosynthetic gene clusters for BLMs from Streptomyces verticillus ATCC15003, tallysomycins from Streptoalloteichus hindustanus E465-94 ATCC31158, and zorbamycin (ZBM) from Streptomyces flavoviridis SB9001. Comparative analysis of the three biosynthetic machineries provided the molecular basis for the formulation of hypotheses to engineer novel analogues. We now report engineered production of three new analogues, 6'-hydroxy-ZBM, BLM Z, and 6'-deoxy-BLM Z and the evaluation of their DNA cleavage activities as a measurement for their potential anticancer activity. Our findings unveiled: (i) the disaccharide moiety plays an important role in the DNA cleavage activity of BLMs and ZBMs, (ii) the ZBM disaccharide significantly enhances the potency of BLM, and (iii) 6'-deoxy-BLM Z represents the most potent BLM analogue known to date. The fact that 6'-deoxy-BLM Z can be produced in reasonable quantities by microbial fermentation should greatly facilitate follow-up mechanistic and preclinical studies to potentially advance this analogue into a clinical drug.

Sensitive DNA-Based Electrochemical Strategy for Trace Bleomycin Detection

Bleomycins (BLMs) are widely used in combination with chemotherapy for the treatment of a variety of cancers. The clinical application of BLMs is featured by the occurrence of sometimes fatal side effects, such as renal and lung toxicity, and the potential dose-limiting side effect of pulmonary fibrosis. Therefore, it is highly desirable to develop a sensitive method to quantitatively determine the BLM content in both pharmaceutical analysis and clinical samples, to make full use of therapeutic efficacy and to weaken its toxicity. Here, we proposed a simple, rapid, and convenient electrochemical assay for trace BLM detection. A reported DNA motif, as substrate for BLMs, is prepared to self-assemble onto the gold electrode to fabricate an electrochemical DNA (E-DNA) sensor, with a terminus tethered on the electrode surface and the other terminus labeled with ferrocenyl moiety as a signal reporter to form a stem-loop structure, giving an arise of remarkable faradaic current. In the presence of Fe(II)·BLM, the E-DNA sensor undergoes the irreversible cleavage event, which can be transduced into a significant decrease in current peak. This proposed sensor reveals an impressive sensitivity as low as 100 pM BLMs and exhibits a good performance as well as in serum sample. Considering the high sensitivity and specificity of this proposed sensor, as well as the cost-effective and simple-to-implement features of the electrochemical technique, we believe that this method shows distinct advantages over conventional methods and it is a promising alternative for the determination of trace amounts of BLMs in clinical samples.

The biosyntheticgene cluster of zorbamycin, a member of the bleomycin family of antitumor antibiotics, from Streptomyces flavoviridis ATCC 21892

The biosynthetic gene cluster for the glycopeptide-derived antitumor antibiotic zorbamycin (ZBM) was cloned by screening a cosmid library of Streptomyces flavoviridis ATCC 21892. Sequence analysis revealed 40 ORFs belonging to the ZBM biosynthetic gene cluster. However, only 23 and 22 ORFs showed striking similarities to the biosynthetic gene clusters for the bleomycins (BLMs) and tallysomycins (TLMs), respectively; the remaining ORFs do not show significant homology to ORFs from the related BLM and TLM clusters. The ZBM gene cluster consists of 16 nonribosomal peptide synthetase (NRPS) genes encoding eight complete NRPS modules, three incomplete didomain NRPS modules, and eight freestanding single NRPS domains or associated enzymes, a polyketide synthase (PKS) gene encoding one PKS module, six sugar biosynthesis genes, as well as genes encoding other biosynthesis and resistance proteins. A genetic system using Escherichia coli-Streptomyces flavoviridis intergeneric conjugation was developed to enable ZBM gene cluster boundary determinations and biosynthetic pathway manipulations.

Identification of Strong DNA Binding Motifs for Bleomycin

The bleomycins (BLMs) are clinically used antitumor antibiotics. Their mechanism of action is believed to involve oxidative cleavage of DNA and possibly also RNA degradation. DNA degradation has been studied extensively and shown to involve binding of an activated metallobleomycin to DNA, followed by abstraction of C4'-H from deoxyribose in the rate-limiting step for DNA degradation. It is interesting that while DNA and RNA degradation by activated Fe.BLM has been studied extensively, much less is known about the actual binding selectivity of BLM, that is, the obligatory step that precedes cleavage. Thus it is unclear whether cleavage specificity is defined by the binding event or whether cleavage occurs at a subset of preferred binding sites. With only a few exceptions, NMR binding studies have employed metalloBLMs such as Co.BLM and Zn.BLM whose therapeutic relevance is uncertain. A single biochemical study that compared DNA binding and cleavage directly also employed Co.BLM. It is logical to anticipate that preferred sites of DNA cleavage will occur at sites that are (a subset of) preferred DNA binding sites, but there are currently no data available relevant to this issue. Herein, we describe the development and implementation of a novel strategy to identify DNA motifs that bind BLM strongly.