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Journal: Bioconjugate chemistry


Cycloxygenase-2 (COX-2) is an attractive target for molecular imaging because it is an inducible enzyme that is expressed in response to inflammatory and proliferative stimuli. Recently, we reported that conjugation of indomethacin with carboxy-X-rhodamine dyes results in the formation of effective, targeted, optical imaging agents able to detect COX-2 in inflammatory tissues and pre-malignant and malignant tumors (Uddin et al. Cancer Res. 2010, 70, 3618-3627). The present paper summarizes the details of the structure-activity relationship (SAR) studies performed for lead optimization of these dyes. A wide range of fluorescent conjugates were designed and synthesized, and each of them was tested for their ability to selectively inhibit COX-2 as the purified protein and in human cancer cells. The SAR study revealed that indomethacin conjugates are the best COX-2-targeted agents compared to the other carboxylic acid-containing non-steroidal anti-inflammatory drugs (NSAIDs) or COX-2-selective inhibitors (COXIBs). An n-butyldiamide linker is optimal for tethering bulky fluorescent functionalities onto the NSAID or COXIB cores. The activity of conjugates also depends on the size, shape, and electronic properties of the organic fluorophores. These reagents are taken up by COX-2-expressing cells in culture, and the uptake is blocked by pretreatment with a COX inhibitor. In in vivo settings, these reagents become highly enriched in COX-2-expressing tumors compared to surrounding normal tissue, and they accumulate selectively in COX-2-expressing tumors as compared with COX-2-negative tumors implanted in the same mice. Thus, COX-2-targeted fluorescent inhibitors are useful for preclinical and clinical detection of lesions containing elevated levels of COX-2.

Concepts: Cancer, Cyclooxygenase, Non-steroidal anti-inflammatory drug, Anti-inflammatory, Paracetamol, Ibuprofen, Diclofenac, Celecoxib


Liposomes are nanocarriers that deliver the payloads at the target site, leading to therapeutic drug concentrations at the diseased site and reduced toxic effects in healthy tissues. Several approaches have been used to enhance the ability of the nanocarrier to target the specific tissues, including ligand-targeted liposomes and stimuli-responsive liposomes. Ligand-targeted liposomes exhibit higher uptake by the target tissue due to the targeting ligand attached to the surface, while, the stimuli-responsive liposomes do not release their cargo unless they expose to an endogenous or exogenous stimulant at the target site. In this review, we mainly focus on the liposomes that are responsive to pathologically increased levels of enzymes at the target site. Enzyme-responsive liposomes release their cargo upon contact with the enzyme through several destabilization mechanisms: a) structural perturbation in the lipid bilayer, b) removal of a shielding polymer from the surface and increased cellular uptake, c) cleavage of a lipopeptide or lipopolymer incorporated in the bilayer, and d) activation of a prodrug in the liposomes.

Concepts: Protein, Medicine, Cancer, Enzyme, Cure, Poison, Doxorubicin, Recreational drug use


Pyridoxal-5'-phosphate (PLP) represents an active form of Vitamin B6 that shows relatively fast imine formation with hydrazines under physiological conditions without the need of a catalyst. A convenient phosphate/amine conjugation protocol was developed to covalently link PLP to proteins, affording proteins capable of hydrazone formation with bioorthogonal hydrazinyl functional groups. Thus, the lectin Concanavalin A (Con A) was labeled with PLP. Pretreatment with fluorescein hydrazide gave dye-labeled Con A that labeled cell surfaces efficiently. Alternatively, pretargeting was achieved by labeling cells with Con A-PLP, then treatment in vitro with Alexa Fluor 488 hydrazide.

Concepts: Proteins, Functional group, Ketone, Functional groups, Aldehyde, Concanavalin A, Lectin, Vitamin B6


The rational design of molecules with selective intracellular targeting is a great challenge of contemporary chemistry and life sciences. Here, we demonstrate rational approach to development of compartment-specific fluorescent dyes from γ-aryl substituted pentamethine family. These novel dyes exhibit an extraordinary affinity and selectivity for cardiolipin in inner mitochondrial membrane and possess excellent photostability, fluorescent properties and low phototoxicity. Selective imaging of live and fixed mitochondria was achieved in various cell lines using nanomolar concentrations of these dyes. Their high localization specificity and low toxicity enables to study morphological changes, structural complexity and dynamics of mitochondria playing pivotal role in many pathological diseases. These far-red emitting dyes could also serve for a variety of biomedical applications.

Concepts: DNA, Oxygen, Cell, Mitochondrion, Organelle, Cell biology, Cytoplasm, Chemistry


The therapeutic application of small interfering RNA (siRNA) requires safe nanocarriers for specific and efficient delivery in vivo. Herein, PEGylated cationic cerasomes (PCCs) were fabricated by doping a cationic lipid with hydroxyl group into nanohybrid cerasomes. Multiple properties of PCCs provide a solution to many of the limitations associated with current delivery platforms of siRNA. The polyorganosiloxane surface imparts PCCs higher morphological stability than conventional liposomes. The PEGylation of the cationic cerasome could protect the cerasome nanoparticles from agglomeration and macrophage capture, reduce protein absorption, consequently prolong the blood circulating time and enhance the siRNA delivery efficiency. In addition, incorporation of the lipid containing hydroxyl group further facilitates endosome release. Moreover, PCCs were further to transport siRNA into the cytosol primarily via endocytosis. When applied to systemic administration, PCCs have demonstrated effective delivery into liver and preferential uptake by hepatocytes in mice, thereby leading to high siRNA gene-silencing activity. All these results show potential therapeutic applications of PCCs mediated delivery of siRNA for liver diseases.

Concepts: Protein, RNA, Messenger RNA, Small interfering RNA, RNA interference, Liver, Bile, Proteomics


Acetylcholinesterase (AChE) is the physiological target of organophosphorus nerve agent compounds. Currently, the development of a formulation for prophylactic administration of cholinesterases as bioscavengers in established risk situations of exposure to nerve agents is the incentive for many efforts. While cholinesterases bioscavengers were found to be highly effective in conferring protection against nerve agent exposure in animal models, their therapeutic use is complicated by short circulatory residence time. To create a bioscavenger with prolonged plasma half-life, compatible with biotechnological production and purification, a chimeric recombinant molecule of HuAChE coupled to the Fc region of human IgG1 was designed. The novel fusion protein, expressed in cultured cells under optimized conditions, maintains its full enzymatic activity, at levels similar to those of the native AChE enzyme. Thus, this novel fusion product retained its bioscavenging reactivity toward the organophosphate-AChE inhibitors BW284c5, propidium, soman and VX. Furthermore, when administered to mice, AChE-Fc exhibits exceptionally circulatory residence longevity (MRT of 6000 min), superior to any other known cholinesterase-based recombinant bioscavengers. Owing to its optimized pharmacokinetic performance, high reactivity toward nerve agents and ease of production, AChE-Fc emerges as a promising next-generation organophosphorus bioscavenger.

Concepts: Protein, Enzyme, Acetylcholine, Sarin, Organophosphate, VX, Nerve agent, Ibn al-Nafis


Liver X receptor (LXR) agonists have been explored as potential treatments for atherosclerosis and other diseases based on their ability to induce reverse cholesterol transport and suppress inflammation. However, this therapeutic potential has been hindered by on-target adverse effects in the liver mediated by excessive lipogenesis. Herein, we report a novel site-specific antibody-drug conjugate (ADC) that selectively delivers a LXR agonist to monocytes/macrophages while sparing hepatocytes. The unnatural amino acid para-acetylphenylalanine (pAcF) was site-specifically incorporated into anti-CD11a IgG, which binds the α-chain component of the lymphocyte function-associated antigen 1 (LFA-1) expressed on nearly all monocytes and macrophages. An aminooxy-modified LXR agonist was conjugated to anti-CD11a IgG through a stable, cathepsin B cleavable oxime linkage to afford a chemically-defined ADC. The anti-CD11a IgG-LXR agonist ADC induced LXR activation specifically in human THP-1 monocyte/macrophage cells in vitro (EC50 ~ 27 nM), but had no effect in hepatocytes, indicating that payload delivery is CD11a-mediated. Moreover, the ADC exhibited higher fold activation compared to a conventional synthetic LXR agonist T0901317 (Tularik) (3 fold). This novel ADC represents a fundamentally different strategy that uses tissue targeting to overcome the limitations of LXR agonists for potential use in treating atherosclerosis.

Concepts: Cholesterol, Immune system, White blood cell, Protein, Atherosclerosis, Liver, Serotonin, Liver X receptor


Activatable cell-penetrating peptides are of great interest in drug delivery because of their enhanced selectivity which can be controlled by the external stimuli that triggers their activation. The use of a specific enzymatic reaction to trigger uptake of an inert peptide offers a relevant targeting strategy because the activation process takes place in a short time and only in areas where the specific cell surface enzyme is present. To this aim, the lysine side chain of Tat peptides was modified with an enzyme-cleavable domain of minimal size. This yielded blocked Tat-peptides which were inactive but that could be activated by co-incubation with the selected enzymes.

Concepts: Protein, Cell, Bacteria, Amino acid, Enzyme, Glucose, Catalysis, Enzymes


Sacituzumab govitecan (IMMU-132) is an antibody-drug conjugate (ADC) made from a humanized anti-Trop-2 monoclonal antibody (hRS7) conjugated with the active metabolite of irinotecan, SN-38. In addition to its further characterization, as the clinical utility of IMMU-132 expands to an ever-widening range of Trop-2-expressing solid tumor types, its efficacy in new disease models needs to be explored in a non-clinical setting. Unlike most ADCs that use ultra-toxic drugs and stable linkers, IMMU-132 uses a moderately toxic drug with a moderately stable carbonate bond between SN-38 and the linker. Flow cytometry and immunohistochemistry disclosed Trop-2 is expressed in a wide range of tumor types, including gastric, pancreatic, triple-negative breast (TNBC), colonic, prostate, and lung. While cell-binding experiments reveal no significant differences between IMMU-132 and parental hRS7 antibody, surface plasmon resonance analysis using a Trop-2 CM5 chip shows a significant binding advantage for IMMU-132 over hRS7. The conjugate retained binding to the neonatal receptor, but lost greater than 60% of the antibody-dependent cell-mediated cytotoxicity activity compared to hRS7. Exposure of tumor cells to either free SN-38 or IMMU-132 demonstrated the same signaling pathways, with pJNK1/2 and p21WAF1/Cip1 up-regulation followed by cleavage of caspases 9, 7, and 3, ultimately leading to poly-ADP-ribose polymerase cleavage and double-stranded DNA breaks. Pharmacokinetics of the intact ADC in mice reveals a mean residence time (MRT) of 15.4 h, while the carrier hRS7 antibody cleared at a similar rate as unconjugated antibody (MRT = ~300 h). IMMU-132 treatment of mice bearing human gastric cancer xenografts (17.5 mg/kg; twice weekly x 4 weeks) resulted in significant anti-tumor effects compared to mice treated with a non-specific control. Clinically relevant dosing schemes of IMMU-132 administered either every other week, weekly, or twice weekly in mice bearing human pancreatic or gastric cancer xenografts demonstrate similar, significant anti-tumor effects in both models. Current Phase I/II clinical trials (, NCT01631552) confirm anticancer activity of IMMU-132 in cancers expressing Trop-2, including gastric and pancreatic cancer patients.

Concepts: Immune system, Cancer, Oncology, Benign tumor, Tumor, Neoplasm, Surface plasmon resonance, Antibody-dependent cell-mediated cytotoxicity


This article reports an effective method to regulate hydrophobic drug release rate from partially silica-coated bicellar nanodisc generated from proamphiphilic organoalkoxysilane and dihexanoyl phosphatidylcholine by introducing different molar percentages of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG2000 (DSPE-PEG2000) into planar bilayers of hybrid bicelles. It was found that the drug release rate increased with increasing the molar percentages of DSPE-PEG2000, and 57.38%, 69.21%, 78.69%, 81.64% and 82.23% of hydrophobic doxorubicin was released within 120 h from the nanodics incorporating with 0%, 2.5%, 5%, 10% and 20% DSPE-PEG2000, respectively. Compared with the non-PEGylated nanodisc and free doxorubicin, the PEGylated nanodiscs showed good biocompatibility, high cellular uptake and adhesion, as well as high local drug accumulation. In addition, both in vitro and in vivo results demonstrated significantly improved antitumor efficacy of the PEGylated nanodisc than its control groups. Thus, the PEGylated nanodisc with partial silica coating offers a facile and efficient strategy of drug delivery for chemotherapy with improved patient acceptance and compliance.

Concepts: Chemotherapy, In vivo, In vitro, Doxorubicin, Release, Phosphatidylcholine