Red-Blood-Cell-Membrane-Coated Metal-Drug Nanoparticles for Enhanced Chemotherapy.

To overcome high toxicity, low bioavailability and poor water solubility of chemotherapeutics, a variety of drug carriers have been designed. However, most carriers are severely limited by low drug loading capacity and adverse side effects. Here, a new type of metal-drug nanoparticles (MDNs) was designed and synthesized. The MDNs self-assembled with Fe(III) ions and drug molecules through coordination, resulting in nanoparticles with high drug loading. To assist systemic delivery and prolong circulation time, the obtained MDNs were camouflaged with red blood cell (RBCs) membranes (RBCs@Fe-DOX MDNs) to improve their stability and dispersity. The RBCs@Fe-DOX MDNs presented pH-responsive release functionalities, resulting in drug release accelerated in acidic tumor microenvironments. The outstanding in vitro and in vivo antitumor therapeutic outcome was realized by RBCs@Fe-DOX MDNs. This study provides an innovative design guideline for chemotherapy and demonstrates the great capacity of nanomaterials in anticancer treatments.

EGFR-targeted prodrug activation using bioorthogonal alkene-azide click-and-release chemistry.

Epidermal growth factor receptor (EGFR) is overexpressed in many cancers and therefore serves as an excellent target for prodrug activation. Functionalised trans-cyclooctenes (TCO) were conjugated to an EGFR antibody (cetuximab), providing a reagent for pre-targeting and localisation of the bioorthogonal reagent. The TCOs react with a 4-azidobenzyl carbamate doxorubicin prodrug via a [3 + 2]-cycloaddition and subsequent self-immolation leads to release of doxorubicin (click-and-release). In vitro cell-based assays demonstrated proof-of-concept, that cetuximab conjugated to highly strained TCO (AB-d-TCO) could bind to the EGFR in a melanoma cell line, and selectively activate the doxorubicin prodrug. In a non-EGFR expressing melanoma cell line, no significant prodrug activation was observed. In vivo experiments using this combination of AB-d-TCO and the azido-doxorubicin prodrug in a murine melanoma model revealed no significant anti-tumour activity or increased survival, suggesting there was insufficient prodrug activation and drug release at the tumour site.

Design and synthesis of a folate-receptor targeted diazepine-ring-opened pyrrolobenzodiazepine prodrug conjugate.

Pyrrolobenzodiazepines (PBDs) and their dimers (bis-PBDs) have emerged as some of the most potent chemotherapeutic compounds and are currently under development as novel payloads in antibody-drug conjugates (ADCs). However, when used as stand-alone therapeutics or as warheads for small molecule drug conjugates (SMDCs), dose-limiting toxicities are often observed. As an elegant solution to this inherent problem, we designed and synthesized a diazepine-ring-opened bis-PBD prodrug (pro-PBD-PBD) folate conjugate lacking the one of the two imine moieties found in the corresponding free bis-PBD. Upon entering a targeted cell, cleavage of the linker system, including the hydrolysis of an oxazolidine moiety, results in the formation of a reactive intermediate which possesses a newly formed aldehyde as well as an aromatic amine. A fast and spontaneous intramolecular ring-closing reaction subsequently takes place as the aromatic amine adds to the aldehyde with the loss of water to give the imine, and as a result, the diazepine ring, thereby delivering the bis-PBD to the targeted cell. The in vitro and in vivo activity of this conjugate has been evaluated on folate receptor positive KB cells. Sub-nanomolar activity with good specificity and high cure rates with minimal toxicity have been observed.

Relationship among structure, cytotoxicity, and Michael acceptor reactivity of quinocidin.

Quinocidin (QCD) is a cytotoxic antibiotic with an unusual 3,4-dihydroquinolizinium skeleton. We previously found that QCD captures thiols in neutral aqueous media via a Michael addition-type reaction. However, it remains unclear whether the Michael acceptor reactivity of QCD is responsible for its cytotoxicity. In this study, we synthesized thirteen analogs of QCD to examine the relationship among its structure, cytotoxicity, and reactivity toward thiols. Thiol-trapping experiments and cytotoxicity tests collectively suggested that the Michael acceptor function of QCD is independent of its cytotoxic activity, and that the pyridinium moiety with the hydrophobic side chain is a key structural factor for cytotoxicity. These findings further led us to demonstrate that incorporation of an amide group into the side chain of QCD significantly reduced its toxicity but hardly affected the Michael acceptor function. The present study lays the foundation for QCD-based drug design and highlights the potential of QCD as a unique electrophile for use in the development of covalent inhibitors and protein-labeling probes.

Androsamide, a Cyclic Tetrapeptide from a Marine Nocardiopsis sp., Suppresses Motility of Colorectal Cancer Cells.

A cyclic tetrapeptide, androsamide (1), was isolated from a marine actinomycete of the genus Nocardiopsis, strain CNT-189. The planar structure of 1 was assigned by the interpretation of 1D and 2D NMR spectroscopic data. The absolute configurations of constituent amino acids of 1 were determined by application of the Marfey's and advanced Marfey's methods. Androsamide (1) strongly suppressed the motility of Caco2 cells caused by epithelial-mesenchymal transition.

Lipid/Hyaluronic Acid-Coated Doxorubicin-Fe3O4 as a Dual-Targeting Nanoparticle for Enhanced Cancer Therapy.

Development of a delivery system to lower systemic toxicity and enhance doxorubicin (DOX) antitumor efficacy against multi-drug resistant (MDR) tumors is of great clinical significance. Here, lipid/hyaluronic acid (HA)-coated DOX-Fe3O4 was characterized to determine its optimal safety and efficacy on a tumor. DOX was first conjugated onto the Fe3O4 NPs surface, which was subsequently coated with phosphatidylcholine (PC) lipids, which consisted of a tumor cell-targeting HA ligand, to generate a dual-targeting nanoparticle (NP). DOX-Fe3O4 synthesis was validated by the Fourier-transform infrared (FT-IR) analysis results. Core-shell PC/HA@DOX-Fe3O4 formation, which had an average particle size of 48.2 nm, was observed based on the transmission electron microscopy (TEM) and dynamic laser light scattering (DLS) results. The saturation magnetization value of PC/HA@DOX-Fe3O4 was discovered to be 28 emu/g using vibrating-sample magnetometry. Furthermore, the designed PC/HA@DOX-Fe3O4 achieved greater MCF-7/ADR cellular uptake and cytotoxicity as compared with DOX. In addition, PC/HA@DOX-Fe3O4 exhibited significant DOX tumor-targeting capabilities and enhanced tumor growth inhibition activity in the xenograft MCF-7/ADR tumor-bearing nude mice following magnetic attraction and ligand-mediated targeting, with less cardiotoxicity. Therefore, PC/HA@DOX-Fe3O4 is a potential candidate for MDR tumor chemotherapy. Graphical abstract.

Programming Drug Delivery Kinetics for Active Burst Release with DNA Toehold Switches.

Despite smart drug delivery systems (DDSs) with high delivery efficiency and improved efficacy for chemotherapy, precise drug release in targeted tumor cells in a controllable way is still challenging. In this work, we develop DNA toehold switch-engineered spherical nucleic acid-templated hydrogel (SNAgel) for on-site active burst release of chemotherapeutic (tumor-killing) drugs in target cancer cells. By designing ligand-specific toehold sequences on hybridization chain reaction (HCR)-generated SNAgel, we realize burst release of payloads with a wide range of t1/2 ranging from 60.52 to 5.49 min by active dynamic control of the kinetics. The camouflage of SNAgels with compact DNA shell enables elongated/prolonged blood circulation and targeted accumulation, cell entry, and apoptosis induction in vivo. The enhanced anticancer activity of SNAgels was substantiated in both cancer cell lines and xenografted tumor-bearing mice. This DNA-engineered kinetic control approach sheds new light on developing paradigm-shifting DDSs for cancer therapeutics.

12b80 - Hydroxybisphosphonate Linked Doxorubicin: Bone Targeted Strategy for Treatment of Osteosarcoma.

To reply to as yet unmet medical needs to treat osteosarcoma, a form of primary bone cancer, we conceived the 12b80 compound by covalently conjugating antineoplastic compound doxorubicin to a bone targeting hydroxybisphosphonate vector and turned it into a prodrug through a custom linker designed to specifically trigger doxorubicin release in acidic bone tumor microenvironment. Synthesis of 12b80 was thoroughly optimized to be produced at gram scale. 12b80 was evaluated in vitro for high bone support affinity, specific release of doxorubicin in acidic condition, lower cytotoxicity, and cellular uptake of the prodrug. In vivo in rodents, 12b80 displayed rapid and sustained targeting of bone tissue and tumor-associated heterotopic bone and permitted a higher doxorubicin payload in tumor bone environment compared to nonvectorized doxorubicin. Consequently, 12b80 showed much lower toxicity compared to doxorubicin, promoted strong antitumor effects on rodent orthotopic osteosarcoma, displayed a dose-response therapeutic effect, and was more potent than doxorubicin/zoledronate combination.

Synthesis and antitumor activity of daunorubicin conjugates with of 3,4-methylendioxybenzaldehyde.

The design of hybrid (chimeric) molecules containing two different pharmacophores connected via a spacer (linker) is a promising approach to the functionalization of natural compounds and potentially of drug molecules. These are important examples for the use of this approach with anthracycline antibiotics. The use of this methodology may help to eliminate some of the drawbacks of anthracycline drugs, e.g., high cardiotoxicity and MDR development.

Design, synthesis and biological evaluation of N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide derivatives as novel P-glycoprotein inhibitors reversing multidrug resistance.

The overexpression of P-glycoprotein plays an important role in the process of multidrug resistance (MDR). P-gp inhibitors are one of the effective strategies to reverse tumor MDR. Novel P-gp inhibitors with phthalazinone scaffolds were designed, synthesized and evaluated. Compound 26 was found to be the most promising for further study. Compound 26 possessed high potency (EC50 = 46.2 ±â€¯3.5 nM) and low cytotoxicity.26 possessed high MDR reversal activity towards doxorubicin-resistant K56/A02 cells. Reversal fold (RF) value reach to 44.26. 26 also increased accumulation of doxorubicin (DOX or ADM) or other MDR-related anticancer drugs with different structures. In conclusion, compound 26 deserves more research for its good features as P-gp inhibitor.

The antitumor antibiotic rebeccamycin-challenges and advanced approaches in production processes.

Rebeccamycin is an antibiotic and antitumor substance isolated from the filamentous bacterium Lentzea aerocolonigenes. After its discovery, investigations of rebeccamycin focused on elucidating its structure, biological activity, and biosynthetic pathway. For potential medical application, a sufficient drug supply has to be ensured, meaning that the production process of rebeccamycin plays a major role. In addition to the natural production of rebeccamycin in L. aerocolonigenes, where the complex cell morphology is an important factor for a sufficient production, rebeccamycin can also be heterologously produced or chemically synthesized. Each of these production processes has its own challenges, and first approaches to production often lead to low final product concentrations, which is why process optimizations are performed. This review provides an overview of the production of rebeccamycin and the different approaches used for rebeccamycin formation including process optimizations.

Synthesis of Myrocin G, the Putative Active Form of the Myrocin Antitumor Antibiotics.

The antiproliferative antimicrobial fungal metabolites known as the myrocins have been proposed to cross-link DNA by double nucleotide addition. However, the nature of the DNA-reactive species is ambiguous, as myrocins have been isolated as functionally distinct 5-hydroxy-γ-lactone and diosphenol isomers. Based on literature precedent, we hypothesized that the diosphenol 7 (assigned here the trivial name myrocin G) is the biologically active form of the representative isolate (+)-myrocin C (1). To probe this, we developed a short enantioselective route to 7. A powerful fragment-coupling reaction that forms the central ring of the target in 38% yield and in a single step was developed. In support of our hypothesis, 7 was efficiently transformed to the bis(sulfide) 6, a product previously isolated from reactions of 1 with excess benzenethiol. This work provides the first direct access to the diosphenol 7, sets the stage for elucidating the mode of interaction of the myrocins with DNA, and provides a foundation for the synthesis of other pimarane diterpenes.

Chemo-enzymatic Total Syntheses of Jorunnamycin A, Saframycin A, and N-Fmoc Saframycin Y3.

The antitumor tetrahydroisoquinoline (THIQ) alkaloids share a common pentacyclic scaffold that is biosynthesized by nonribosomal peptide synthetases involving unique enzymatic Pictet-Spengler cyclizations. Herein we report concise and divergent chemo-enzymatic total syntheses of THIQ alkaloids by merging precise chemical synthesis with in vitro engineered biosynthesis. A recombinant enzyme SfmC responsible for the biosynthesis of saframycin A was adapted for the assembly of these natural products and their derivatives, by optimizing designer substrates compatible with SfmC through chemical synthesis. The appropriately functionalized pentacyclic skeleton were efficiently synthesized by streamlining the linkage between SfmC-catalyzed multistep enzymatic conversions and chemical manipulations of the intermediates to install aminonitrile and N-methyl groups. This approach allowed rapid access to the elaborated pentacyclic skeleton in a single day starting from two simple synthetic substrates without isolation of the intermediates. Further functional group manipulations allowed operationally simple and expeditious syntheses of jorunnamycin A, saframycin A, and N-Fmoc saframycin Y3 that could be versatile and common precursors for the artificial production of other antitumor THIQ alkaloids and their variants.

Streamlined Total Synthesis of Shishijimicin A and Its Application to the Design, Synthesis, and Biological Evaluation of Analogues thereof and Practical Syntheses of PhthNSSMe and Related Sulfenylating Reagents.

Shishijimicin A is a scarce marine natural product with highly potent cytotoxicities, making it a potential payload or a lead compound for designed antibody-drug conjugates. Herein, we describe an improved total synthesis of shishijimicin A and the design, synthesis, and biological evaluation of a series of analogues. Equipped with appropriate functionalities for linker attachment, a number of these analogues exhibited extremely potent cytotoxicities for the intended purposes. The synthetic strategies and tactics developed and employed in these studies included improved preparation of previously known and new sulfenylating reagents such as PhthNSSMe and related compounds.

The Mechanism of Action of (-)-Lomaiviticin A.

(-)-Lomaiviticin A (4) is a complex C2-symmetric bacterial metabolite that contains two diazofluorene functional groups. The diazofluorene consists of naphthoquinone, cyclopentadiene, and diazo substituents fused through a σ- and π-bonding network. Additionally, (-)-lomaiviticin A (4) is a potent cytotoxin, with half-maximal inhibitory potency (IC50) values in the low nanomolar range against many cancer cell lines. Because of limitations in supply, its mechanism of action had remained a "black box" since its isolation in the early 2000s. In this Account, I describe how studies directed toward the total synthesis of (-)-lomaiviticin A (4) provided a platform to elucidate the emergent properties of this metabolite and thereby connect chemical reactivity with cellular phenotype. We first developed a convergent strategy to prepare the diazofluorene (9 + 10 → 13). We then adapted this chemistry to the synthesis of lomaiviticin aglycon (21/22) and the natural monomeric diazofluorene (-)-kinamycin F (3). The key step in the lomaiviticin aglycon (21/22) synthesis involved the stereoselective oxidative coupling of two monomeric diazofluorenes (2 × 18 → 20) to establish the cojoining carbon-carbon bond of the target. As the absolute stereochemistry of the aglycon and carbohydrate residues of (-)-lomaiviticin A (4) were unknown, we developed a semisynthetic route to the metabolite that proceeds in one step and 42% yield by diazo transfer to the more abundant isolate (-)-lomaiviticin C (6). This allowed us to complete the stereochemical assignment of (-)-lomaiviticin A (4) and provided a renewable source of material. Using this material, we established that the remarkable cytotoxic effects of (-)-lomaiviticin A (4) derive from the induction of highly toxic double-strand breaks (DSBs) in DNA. At the molecular level, 1,7-nucleophilic additions to each electrophilic diazofluorene trigger homolytic decomposition pathways that produce sp2 radicals at the carbon atoms of each diazo group. These radicals abstract hydrogen atoms from the deoxyribose of DNA, a process known to initiate strand cleavage. NMR spectroscopy and molecular mechanics simulations were used to elucidate the mode of DNA binding. These studies showed that both diazofluorenes of (-)-lomaiviticin A (4) penetrate into the duplex. This mode of non-covalent binding places each diazo carbon atom in close proximity to each DNA strand. Throughout these studies, isolates containing one diazofluorene, such as (-)-lomaiviticin C (6) and (-)-kinamycin C (2), were used as controls. Consistent with our mechanistic model, these compounds do not induce DSBs in DNA and are several orders of magnitude less potent. Reactivity studies suggest that (-)-lomaiviticin A (4) is more electrophilic than simple monomeric diazofluorenes. We attribute this to through-space delocalization of the developing negative charge in the transition state for 1,7-addition. Consistent with this mechanism of action, (-)-lomaiviticin A (4) displays selective low-picomolar potencies toward DNA DSB repair-deficient cell types. The emergent properties of (-)-lomaiviticin A (4) derive from the specific arrangement of diazo, naphthoquinone, cyclopentadiene, and ketone functional groups. These functional groups work together to yield, essentially, a masked vinyl radical that can be exposed under biological conditions. Furthermore, the rotational symmetry of the metabolite, deriving from dimerization, allows it to interact with the antiparallel symmetry of DNA and affect cleavage of the duplex.

Synthesis and biological evaluation of novel doxorubicin-containing ASGP-R-targeted drug-conjugates.

Asialoglycoprotein receptor (ASGP-R) belongs to a wide family of C-type lectins and it is currently regarded as an attractive protein in the field of targeted drug delivery (TDD). It is abundantly expressed in hepatocytes and can be found predominantly on the sinusoidal surface especially of HepG2 cells. Therefore, ASGP-R can be used for the TDD of anticancer therapeutics against HCC and molecular diagnostic tools. To date, a variety of mono- and multivalent selective ASGP-R ligands have been discovered. Although many of these compounds have demonstrated a relatively high binding affinity towards the target, the reported synthetic schemes are not handled, complicated and include many non-trivial steps. In the current study, we describe a convenient and versatile synthetic approach to novel monovalent drug-conjugates containing N-acetyl-2-deoxy-2-aminogalactopyranose fragment as an ASGP-R-recognition "core-head" and well-known nonselective cytostatic - Doxorubicin (Dox). This is the first example of the direct conjugation of a drug molecule to the ASGP-targeted warhead by a really convenient manner via a simple linker sequence. The performed MTS-based biological evaluation in HepG2 cells revealed the novel conjugates as having anticancer activity. Confocal microscopy showed that the molecules readily penetrated HepG2 membrane and were mainly localized within the cytoplasm instead of the nucleus. Per contra, Dox under the same conditions demonstrated good anticancer activity and was predominantly concentrated in the nucleus. Therefore, we speculate that the amide "trigger" that we have used in this study for linker attachment is a sufficiently stable inside the cells to be enzymatically or spontaneously degraded. As a consequence, we did not observe the release of the drug. Ligands containing triggers that are more liable towards endogenous hydrolysis within the tissue of targeting are strongly required.

Chitosan Immobilization on Bio-MOF Nanostructures: A Biocompatible pH-Responsive Nanocarrier for Doxorubicin Release on MCF-7 Cell Lines of Human Breast Cancer.

In this work, a bio-metal-organic framework (Bio-MOF) coated with a monodispersed layer of chitosan (CS; CS/Bio-MOF) was synthesized and applied as a pH-responsive and target-selective system for delivery of doxorubicin (DOX) in the treatment of breast cancer. The efficiency of the nanocarrier in loading and releasing DOX was assessed at different pH levels. To monitor the in vitro drug release behavior of the drug-loaded carrier, the carrier was immersed in a phosphate buffered saline solution (PBS, pH 7.4) at 37 °C. According to the observations, the nanocarrier presents a slow and continuous release profile as well as a noticeable drug loading capacity. In addition, the carrier demonstrates a pH-responsive and target-selective behavior by releasing a high amount of DOX at pH 6.8, which is indicative of tumor cells and inflamed tissues and releasing a substantially lower amount of DOX at higher pH values. In addition, the results indicated that pH is effective on DOX uptake by CS/Bio-MOF. A 3.6 mg amount of DOX was loaded into 10 mg of CS/Bio-MOF, resulting in a 21.7% removal at pH 7.4 and 93.0% at pH 6.8. The collapsing and swelling of the CS layers coated on the surface of the Bio-MOFs were found to be responsible for the observed pH dependence of DOX delivery. Moreover, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the trypan blue test were performed on the MCF-7 (breast cancer) cell line in the presence of the CS/Bio-MOF carrier to confirm its biological compatibility. In addition, Annexin V staining was conducted to evaluate the cytotoxicity of the free and loaded DOX molecules. On the basis of the obtained optical microscopy, MTT assay, fluorescence microscopy, and dyeing results, the CS/Bio-MOF carrier greatly enhances cellular uptake of the drug by the MCF-7 cells and, therefore, apoptosis of the cells due to its biocompatibility and pH-responsive behavior.

Staurosporine Derivatives Generated by Pathway Engineering in a Heterologous Host and Their Cytotoxic Selectivity.

Two new staurosporine derivatives, staurosporines M1 and M2 (4 and 5), in addition to five previously reported metabolites (1-3, 6, and 7), were generated by the heterologous expression of engineered spc gene clusters in Streptomyces coelicolor M1146. The structures of these derivatives were determined by a combination of spectroscopic methods and CD measurement. Compounds 1, 2, 4, and 5 showed effective activities against three tumor cell lines (HCT-116, K562, and Huh 7.5), and 3 was active against HCT-116 and K562 cells. In addition, compounds 3 and 5 showed undetectable toxicity up to 100 µM toward the normal hepatic cell line LO2. Based on the IC50 values, their structure and activity relationships are discussed.

Chemical Activation in Blood Serum and Human Cell Culture: Improved Ruthenium Complex for Catalytic Uncaging of Alloc-Protected Amines.

Chemical (as opposed to light-induced) activation of caged molecules is a rapidly advancing approach to trigger biological processes. We previously introduced the ruthenium-catalyzed release of allyloxycarbonyl (alloc)-protected amines in human cells. A restriction of this and all other methods is the limited lifetime of the catalyst, thus hampering meaningful applications. In this study, we addressed this problem with the development of a new generation of ruthenium complexes for the uncaging of alloc-protected amines with superior catalytic activity. Under biologically relevant conditions, we achieved a turnover number >300, a reaction rate of 580 m-1 s-1 , and we observed high activity in blood serum. Furthermore, alloc-protected doxorubicin, as an anticancer prodrug, could be activated in human cell culture and induced apoptosis with a single low dose (1 µm) of the new catalyst.

Quinocidin, a Cytotoxic Antibiotic with an Unusual 3,4-Dihydroquinolizinium Ring and Michael Acceptor Reactivity toward Thiols.

Cytotoxicity-guided fractionation of the culture broth of Actinomadura sp. TP-A0019 led to the isolation of quinocidin (1), a cytotoxic antibiotic with an unusual 3,4-dihydroquinolizinium ring. The structural assignment was made on the basis of high-field NMR experiments and chemical synthesis. Comparison of the spectral properties of 1 with those of its synthetic counterparts revealed that 1 is a racemic mixture of two enantiomers, which showed similar cytotoxicity against HeLa-S3 cells. Nucleophile-trapping experiments demonstrated that 1 captured 2-mercaptoethanol and N-acetyl-l-cysteine by means of a Michael addition-type reaction, but was inert toward 2-aminoethanol and glycolic acid. Notably, the addition of 1 to thiols proceeded smoothly in neutral aqueous media at room temperature. In view of the thiol-trapping ability and the unusual structure, 1 provides a unique scaffold for designing drug leads and protein-labeling probes.