A comprehensive overview of exosomes as drug delivery vehicles — Endogenous nanocarriers for targeted cancer therapy

Exosomes denote a class of secreted nanoparticles defined by size, surface protein and lipid composition, and the ability to carry RNA and proteins. They are important mediators of intercellular communication and regulators of the cellular niche, and their altered characteristics in many diseases, such as cancer, suggest them to be important both for diagnostic and therapeutic purposes, prompting the idea of using exosomes as drug delivery vehicles, especially for gene therapy. This review covers the current status of evidence presented in the field of exosome-based drug delivery systems. Components for successful exosome-based drug delivery, such as choice of donor cell, therapeutic cargo, use of targeting peptide, loading method and administration route are highlighted and discussed with a general focus pertaining to the results obtained in models of different cancer types. In addition, completed and on-going clinical trials are described, evaluating exosome-based therapies for the treatment of different cancer types. Due to their endogenous origin, exosome-based drug delivery systems may have advantages in the treatment of cancer, but their design needs further refinement to justify their usage on the clinical scale.     

Intracellular cholesterol transporters and modulation of hepatic lipid metabolism: Implications for diabetic dyslipidaemia and steatosis

Aims/hypotheses

To examine hepatic expression of cholesterol-trafficking proteins, mitochondrial StarD1 and endosomal StarD3, and their relationship with dyslipidaemia and steatosis in Zucker (fa/fa) genetically obese rats, and to explore their functional role in lipid metabolism in rat McArdle RH-7777 hepatoma cells.

Methods

Expression of StarD1 and StarD3 in rat liver and hepatoma samples were determined by Q-PCR and/or immunoblotting; lipid mass by colorimetric assays; radiolabelled precursors were utilised to measure lipid synthesis and secretion, and lipidation of exogenous apolipoprotein A-I.

Results

Hepatic expression of StarD3 protein was repressed by genetic obesity in (fa/fa) Zucker rats, compared with lean (Fa/?) controls, suggesting a link with storage or export of lipids from the liver. Overexpression of StarD1 and StarD3, and knockdown of StarD3, in rat hepatoma cells, revealed differential effects on lipid metabolism. Overexpression of StarD1 increased utilisation of exogenous (preformed) fatty acids for triacylglycerol synthesis and secretion, but impacted minimally on cholesterol homeostasis. By contrast, overexpression of StarD3 increased lipidation of exogenous apoA-I, and facilitated de novo biosynthetic pathways for neutral lipids, potentiating triacylglycerol accumulation but possibly offering protection against lipotoxicity. Finally, StarD3 overexpression altered expression of genes which impact variously on hepatic insulin resistance, inducing Ppargcla, Cyp2e1, Nr1h4, G6pc and Irs1, and repressing expression of Scl2a1, Igfbp1, Casp3 and Serpine 1.

Conclusions/interpretation

Targeting StarD3 may increase circulating levels of HDL and protect the liver against lipotoxicity; loss of hepatic expression of this protein, induced by genetic obesity, may contribute to the pathogenesis of dyslipidaemia and steatosis.     

A study in molecular contingency: glutamine phosphoribosylpyrophosphate amidotransferase is a promiscuous and evolvable phosphoribosylanthranilate isomerase

The prevalence of paralogous enzymes implies that novel catalytic functions can evolve on preexisting protein scaffolds. The weak secondary activities of proteins, which reflect catalytic promiscuity and substrate ambiguity, are plausible starting points for this evolutionary process. In this study, we observed the emergence of a new enzyme from the ASKA (A Complete Set of E. coli K-12 ORF Archive) collection of Escherichia coli open reading frames. The overexpression of (His)₆-tagged glutamine phosphoribosylpyrophosphate amidotransferase (PurF) unexpectedly rescued a ΔtrpF E. coli strain from starvation on minimal media. The wild-type PurF and TrpF enzymes are unrelated in sequence, tertiary structure and catalytic mechanism. The promiscuous phosphoribosylanthranilate isomerase activity of the ASKA PurF variant apparently stems from a preexisting affinity for phosphoribosylated substrates. The relative fitness of the (His)₆-PurF/ΔtrpF strain was improved 4.8-fold to nearly wild-type levels by random mutagenesis of purF and genetic selection. The evolved and ancestral PurF proteins were purified and reacted with phosphoribosylanthranilate in vitro. The best evolvant (k_cat/K_M = 0.3 s^− 1 M^− 1) was ∼ 25-fold more efficient than its ancestor but > 10⁷-fold less efficient than the wild-type phosphoribosylanthranilate isomerase. These observations demonstrate in quantitative terms that the weak secondary activities of promiscuous enzymes can dramatically improve the fitness of contemporary organisms.     

Crystal structure of visfatin/pre-B cell colony-enhancing factor 1/nicotinamide phosphoribosyltransferase, free and in complex with the anti-cancer agent FK-866

Visfatin/pre-B cell colony-enhancing factor 1 (PBEF)/nicotinamide phosphoribosyltransferase (NAmPRTase) is a multifunctional protein having phosphoribosyltransferase, cytokine and adipokine activities. Originally isolated as a cytokine promoting the differentiation of B cell precursors, it was recently suggested to act as an insulin analog via the insulin receptor. Here, we describe the first crystal structure of visfatin in three different forms: apo and in complex with either nicotinamide mononucleotide (NMN) or the NAmPRTase inhibitor FK-866 which was developed as an anti-cancer agent, interferes with NAD biosynthesis, showing a particularly high specificity for NAmPRTase. The crystal structures of the complexes with either NMN or FK-866 show that the enzymatic active site of visfatin is optimized for nicotinamide binding and that the nicotinamide-binding site is important for inhibition by FK-866. Interestingly, visfatin mimics insulin signaling by binding to the insulin receptor with an affinity similar to that of insulin and does not share the binding site with insulin on the insulin receptor. To predict binding sites, the potential interaction patches of visfatin and the L1-CR-L2 domain of insulin receptor were generated and analyzed. Although the relationship between the insulin-mimetic property and the enzymatic function of visfatin has not been clearly established, our structures raise the intriguing possibility that the glucose metabolism and the NAD biosynthesis are linked by visfatin.     

Recent advancements in the development of anti-tuberculosis drugs

Modern chemotherapy has significantly improved patient outcomes against drug-sensitive tuberculosis. However, the rapid emergence of drug-resistant tuberculosis, together with the bacterium’s ability to persist and remain latent present a major public health challenge. To overcome this problem, research into novel anti-tuberculosis targets and drug candidates is thus of paramount importance. This review article provides an overview of tuberculosis highlighting the recent advances and tools that are employed in the field of anti-tuberculosis drug discovery. The predominant focus is on anti-tuberculosis agents that are currently in the pipeline, i.e. clinical trials.     

The PRT protein family

Members of the homologous PRT family are catalytic and regulatory proteins involved in nucleotide synthesis and salvage. New crystal structures have revealed key elements of PRT protein function, as well as glimpses of how the fold has evolved to perform both catalytic and regulatory functions.     

Determination of pyrimidine phosphoribosyltransferase and uridine kinase activities by an assay with DEAE-paper discs

Rapid (6 min washing step) and reliable methods for determining uridine kinase and pyrimidine phosphoribosyltransferase activities were devised. These procedures, consisting of a spotting technique on DEAE-discs followed by washing and elution, permitted the consistent recovery of about 90% of the nucleoside 5'-phosphate esters formed from radioactive precursors, either uridine or 5-fluorouracil, respectively. Of these precursors, less than 2% were retained on the discs. Direct counting of the discs (without elution), filtration by either gravity or suction, and the use of so-called activation of discs have not proved advantageous.     

Glutamine PRPP amidotransferase: snapshots of an enzyme in action

Recent studies of glutamine PRPP amidotransferase have provided a new understanding of the function and mechanism of this rather complicated enzyme that may be a paradigm for other complex enzymes. New insights have been gained into the mechanisms of catalysis in the active sites of the two half-reactions, catalytic coupling, allosteric control by feedback inhibitors and the channeling of reaction and metabolic intermediates.     

Distinct mechanisms of hypoxanthine and inosine transport in membrane vesicles isolated from Chinese hamster ovary and Balb 3T3 cells

Both enzyme-mediated group translocation and facilitated diffusion have been proposed as mechanisms by which mammalian cells take up purine bases and nucleosides. We have investigated the mechanisms for hypoxanthine and inosine transport by using membrane vesicles from Chinese hamster ovary cells (CHO), Balb/c 3T3 and SV3T3 cells prepared by identical procedures. Uptake mechanisms were characterized by analyzing intravesicular contents, determining which substrates could exchange with the transport products, assaying for hypoxanthine phosphoribosyltransferase activity, and measuring the stimulation of uptake of hypoxanthine by phosphoribosyl pyrophosphate ($\text{P}\text{Rib}\text{-PP}$).We found that the uptake of hypoxanthine in Balb 3T3 vesicles was stimulated 3–4-fold by $\text{P}\text{Rib}\text{-PP}$. The intravesicular product was predominantly IMP. The hypoxanthine phosphoribosyltransferase activity copurified with the vesicle preparation. These results suggest the possible involvement of this enzyme in hypoxanthine uptake in 3T3 vesicles. In contrast to the 3T3 vesicles, CHO vesicles prepared under identical procedures did not retain hypoxanthine phosphoribosyltransferase activity and did not demonstrate $\text{P}\text{Rib}\text{-PP}$-stimulated hypoxanthine uptake. The intravesicular product of hypoxanthine uptake in CHO vesicles was hypoxanthine. These results and data from our kinetic and exchange studies indicated that CHO vesicles transport hypoxanthine via facilitated diffusion. An analogous situation was observed for inosine uptake; CHO vesicles accumulated inosine via a facilitated diffusion mechanism, while in the same experiments SV3T3 vesicles exhibited a purine nucleoside phosphorylase-dependent translocation of the ribose moiety of inosine.