Mutants and molecular dockings reveal that the primary L-thyroxine binding site in human serum albumin is not the one which can cause familial dysalbuminemic hyperthyroxinemia.

BACKGROUND: Natural mutations of R218 in human serum albumin (HSA) result in an increased affinity for L-thyroxine and lead to the autosomal dominant condition of familial dysalbuminemic hyperthyroxinemia. METHODS: Binding was studied by equilibrium dialysis and computer modeling. RESULTS: Ten of 32 other isoforms tested had modified high-affinity hormone binding. L-thyroxine has been reported to bind to four sites (Tr) in HSA; Tr1 and Tr4 are placed in the N-terminal and C-terminal part of the protein, respectively. Site-directed mutagenesis gave new information about all the sites. CONCLUSIONS: It is widely assumed that Tr1 is the primary hormone site, and that this site, on a modified form, is responsible for the above syndrome, but the binding experiments with the genetic variants and displacement studies with marker ligands indicated that the primary site is Tr4. This new assignment of the high-affinity site was strongly supported by results of MM-PBSA analyses and by molecular docking performed on relaxed protein structure. However, dockings also revealed that mutating R218 for a smaller amino acid increases the affinity of Tr1 to such an extent that it can become the high-affinity site. GENERAL SIGNIFICANCE: Placing the high-affinity binding site (Tr4) and the one which can result in familial dysalbuminemic hyperthyroxinemia (Tr1) in two very different parts of HSA is not trivial, because in this way persons with and without the syndrome can have different types of interactions, and thereby complications, when given albumin-bound drugs. The molecular information is also useful when designing drugs based on L-thyroxine analogues.

Hyaluronic Acid Molecular Weight-Dependent Modulation of Mucin Nanostructure for Potential Mucosal Therapeutic Applications.

This study investigates the effects of different molecular weight hyaluronic acids (HAs) on the mucosal nanostructure using a pig stomach mucin hydrogel as a mucosal barrier model. Microparticles (1.0 µm) and nanoparticles (200 nm) were used as probes, and their movement in mucin was studied by a three-dimensional confocal microscopy-based particle tracking technique and by Nanoparticle Tracking Analysis (NTA) after addition of high-molecular weight (900 kDa) and low-molecular weight (33 kDa) HA. This demonstrated a molecular weight-dependent HA modulation of the mucin nanostructure with a 2.5-fold decrease in the mobility of 200 nm nanoparticles. To further investigate these mechanisms and to verify that the natural viscoelastic properties of mucus are not undesirably altered, rheological measurements were performed on mucin hydrogels with or without HA. This suggested the observed particle mobility restriction was not attributed to alterations of the natural mucin cohesive and viscoelastic properties but, instead, indicates that the added high-molecular weight HA primarily modulates the mucin nanostructure and mesh size. This study, hereby, demonstrates how mucus nanostructure can be modulated by the addition of high-molecular weight HA that offers an opportunity to control mucosal pathogenesis and drug delivery.

Could drugs inhibiting the mevalonate pathway also target cancer stem cells?

Understanding the connection between metabolic pathways and cancer is very important for the development of new therapeutic approaches based on regulatory enzymes in pathways associated with tumorigenesis. The mevalonate cascade and its rate-liming enzyme HMG CoA-reductase has recently drawn the attention of cancer researchers because strong evidences arising mostly from epidemiologic studies, show that it could promote transformation. Hence, these studies pinpoint HMG CoA-reductase as a candidate proto-oncogene. Several recent epidemiological studies, in different populations, have proven that statins are beneficial for the treatment-outcome of various cancers, and may improve common cancer therapy strategies involving alkylating agents, and antimetabolites. Cancer stem cells/cancer initiating cells (CSC) are key to cancer progression and metastasis. Therefore, in the current review we address the different effects of statins on cancer stem cells. The mevalonate cascade is among the most pleiotropic, and highly interconnected signaling pathways. Through G-protein-coupled receptors (GRCP), it integrates extra-, and intracellular signals. The mevalonate pathway is implicated in cell stemness, cell proliferation, and organ size regulation through the Hippo pathway (e.g. Yap/Taz signaling axis). This pathway is a prime preventive target through the administration of statins for the prophylaxis of obesity-related cardiovascular diseases. Its prominent role in regulation of cell growth and stemness also invokes its role in cancer development and progression. The mevalonate pathway affects cancer metastasis in several ways by: (i) affecting epithelial-to-mesenchymal transition (EMT), (ii) affecting remodeling of the cytoskeleton as well as cell motility, (iii) affecting cell polarity (non-canonical Wnt/planar pathway), and (iv) modulation of mesenchymal-to-epithelial transition (MET). Herein we provide an overview of the mevalonate signaling network. We then briefly highlight diverse functions of various elements of this mevalonate pathway. We further discuss in detail the role of elements of the mevalonate cascade in stemness, carcinogenesis, cancer progression, metastasis and maintenance of cancer stem cells.

An Albumin-Oligonucleotide Assembly for Potential Combinatorial Drug Delivery and Half-Life Extension Applications.

The long blood circulatory property of human serum albumin, due to engagement with the cellular recycling neonatal Fc receptor (FcRn), is an attractive drug half-life extension enabling technology. This work describes a novel site-specific albumin double-stranded (ds) DNA assembly approach, in which the 3' or 5' end maleimide-derivatized oligodeoxynucleotides are conjugated to albumin cysteine at position 34 (cys34) and annealed with complementary strands to allow single site-specific protein modification with functionalized ds oligodeoxynucleotides. Electrophoretic gel shift assays demonstrated successful annealing of complementary strands bearing Atto488, 6-carboxyfluorescein (6-FAM), or a factor IXa aptamer to the albumin-oligodeoxynucleotide conjugate. A fluorometric factor IXa activity assay showed retained aptamer inhibitory activity upon assembly with the albumin and completely blocked factor IXa at a concentration of 100 nM for 2 hr. The assembled construct exhibited stability in serum-containing buffer and FcRn engagement that could be increased using an albumin variant engineered for higher FcRn affinity. This work presents a novel albumin-oligodeoxynucleotide assembly technology platform that offers potential combinatorial drug delivery and half-life extension applications.

An albumin-mediated cholesterol design-based strategy for tuning siRNA pharmacokinetics and gene silencing.

Major challenges for the clinical translation of small interfering RNA (siRNA) include overcoming the poor plasma half-life, site-specific delivery and modulation of gene silencing. In this work, we exploit the intrinsic transport properties of human serum albumin to tune the blood circulatory half-life, hepatic accumulation and gene silencing; based on the number of siRNA cholesteryl modifications. We demonstrate by a gel shift assay a strong and specific affinity of recombinant human serum albumin (rHSA) towards cholesteryl-modified siRNA (Kd>1×10(-7)M) dependent on number of modifications. The rHSA/siRNA complex exhibited reduced nuclease degradation and reduced induction of TNF-α production by human peripheral blood mononuclear cells. The increased solubility of heavily cholesteryl modified siRNA in the presence of rHSA facilitated duplex annealing and consequent interaction that allowed in vivo studies using multiple cholesteryl modifications. A structural-activity-based screen of in vitro EGFP-silencing was used to select optimal siRNA designs containing cholesteryl modifications within the sense strand that were used for in vivo studies. We demonstrate plasma half-life extension in NMRI mice from t1/2 12min (naked) to t1/2 45min (single cholesteryl) and t1/2 71min (double cholesteryl) using fluorescent live bioimaging. The biodistribution showed increased accumulation in the liver for the double cholesteryl modified siRNA that correlated with an increase in hepatic Factor VII gene silencing of 28% (rHSA/siRNA) compared to 4% (naked siRNA) 6days post-injection. This work presents a novel albumin-mediated cholesteryl design-based strategy for tuning pharmacokinetics and systemic gene silencing.

Extended blood circulation and joint accumulation of a p(HPMA-co-AzMA)-based nanoconjugate in a murine model of rheumatoid arthritis.

BACKGROUND: We recently synthesized a hydrophilic polymer, poly(N-(2-hydroxypropyl)methacrylamide-co-N-(3-azidopropyl)methacrylamide), p(HPMA-co-AzMA), by RAFT polymerization using a novel azide-containing methacrylamide monomer that through a post modification strategy using click chemistry enabled facile preparation of a panel of versatile and well-defined bioconjugates. In this work we screen a panel of different molecular weight (Mw) fluorescently tagged p(HPMA-co-AzMA) in healthy mice, by live bioimaging, to select an extended circulatory half-life material for investigating joint accumulation in a murine collagen antibody-induced arthritis model. FINDINGS: Fluorescence image analysis revealed half-lifes of <20 min, 2.8 h and 6.4 h for p(HPMA-co-AzMA) of 15, 36 and 54 kDa, respectively, with ~10% polymer retained in the blood after 24 h for the highest Mw. p(HPMA-co-AzMA) of 54 kDa showed enhanced accumulation in the joints of the arthritic mouse model with a bioavailability (AUC = 1783% · h) ~12 times higher (P = 0.01) than healthy control (AUC = 148% · h). CONCLUSIONS: p(HPMA-co-AzMA) of 54 kDa exhibited extended circulatory half-life and preferential accumulation in inflamed joints of a murine model of rheumatoid arthritis (RA). This combined with well-defined polymer size and versatility for conjugation of a range of biomolecules promotes p(HPMA-co-AzMA) for potential applications in the delivery of drugs for treatment of RA.