Bioorthogonal Catalysis for Treatment of Solid Tumors Using Thermostable, Self-Assembling, Single Enzyme Nanoparticles and Natural Product Conversion with Indole-3-acetic Acid.

Bioorthogonal catalysis (BC) generates chemical reactions not present in normal physiology for the purpose of disease treatment. Because BC catalytically produces the desired therapy only at the site of disease, it holds the promise of site-specific treatment with little or no systemic exposure or side effects. Transition metals are typically used as catalytic centers in BC; however, solubility and substrate specificity typically necessitate a coordinating enzyme and/or stabilizing superstructure for in vivo application. We report the use of self-assembling, porous exoshells (tESs) to encapsulate and deliver an iron-containing reaction center for the treatment of breast cancer. The catalytic center is paired with indole-3-acetic acid (IAA), a natural product found in edible plants, which undergoes oxidative decarboxylation, via reduction of iron(III) to iron(II), to produce free radicals and bioactive metabolites. The tES encapsulation is critical for endocytic uptake of BC reaction centers and, when followed by administration of IAA, results in apoptosis of MDA-MB-231 triple negative cancer cells and complete regression of in vivo orthotopic xenograft tumors (p < 0.001, n = 8 per group). When Renilla luciferase (rLuc) is substituted for horseradish peroxidase (HRP), whole animal luminometry can be used to monitor in vivo activity.

A general approach to protein folding using thermostable exoshells.

In vitro protein folding is a complex process which often results in protein aggregation, low yields and low specific activity. Here we report the use of nanoscale exoshells (tES) to provide complementary nanoenvironments for the folding and release of 12 highly diverse protein substrates ranging from small protein toxins to human albumin, a dimeric protein (alkaline phosphatase), a trimeric ion channel (Omp2a) and the tetrameric tumor suppressor, p53. These proteins represent a unique diversity in size, volume, disulfide linkages, isoelectric point and multi versus monomeric nature of their functional units. Protein encapsulation within tES increased crude soluble yield (3-fold to >100-fold), functional yield (2-fold to >100-fold) and specific activity (3-fold to >100-fold) for all the proteins tested. The average soluble yield was 6.5 mg/100 mg of tES with charge complementation between the tES internal cavity and the protein substrate being the primary determinant of functional folding. Our results confirm the importance of nanoscale electrostatic effects and provide a solution for folding proteins in vitro.

Redefining IL11 as a regeneration-limiting hepatotoxin and therapeutic target in acetaminophen-induced liver injury.

Acetaminophen (N-acetyl-p-aminophenol; APAP) toxicity is a common cause of liver damage. In the mouse model of APAP-induced liver injury (AILI), interleukin 11 (IL11) is highly up-regulated and administration of recombinant human IL11 (rhIL11) has been shown to be protective. Here, we demonstrate that the beneficial effect of rhIL11 in the mouse model of AILI is due to its inhibition of endogenous mouse IL11 activity. Our results show that species-matched IL11 behaves like a hepatotoxin. IL11 secreted from APAP-damaged human and mouse hepatocytes triggered an autocrine loop of NADPH oxidase 4 (NOX4)-dependent cell death, which occurred downstream of APAP-initiated mitochondrial dysfunction. Hepatocyte-specific deletion of Il11 receptor subunit alpha chain 1 (Il11ra1) in adult mice protected against AILI despite normal APAP metabolism and glutathione (GSH) depletion. Mice with germline deletion of Il11 were also protected from AILI, and deletion of Il1ra1 or Il11 was associated with reduced c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) activation and quickly restored GSH concentrations. Administration of a neutralizing IL11RA antibody reduced AILI in mice across genetic backgrounds and promoted survival when administered up to 10 hours after APAP. Inhibition of IL11 signaling was associated with the up-regulation of markers of liver regenerations: cyclins and proliferating cell nuclear antigen (PCNA) as well as with phosphorylation of retinoblastoma protein (RB) 24 hours after AILI. Our data suggest that species-matched IL11 is a hepatotoxin and that IL11 signaling might be an effective therapeutic target for APAP-induced liver damage.

In vivo prevention of arterial restenosis with paclitaxel-encapsulated targeted lipid-polymeric nanoparticles.

Following recent successes with percutaneous coronary intervention (PCI) for treating coronary artery disease (CAD), many challenges remain. In particular, mechanical injury from the procedure results in extensive endothelial denudation, exposing the underlying collagen IV-rich basal lamina, which promotes both intravascular thrombosis and smooth muscle proliferation. Previously, we reported the engineering of collagen IV-targeting nanoparticles (NPs) and demonstrated their preferential localization to sites of arterial injury. Here, we develop a systemically administered, targeted NP system to deliver an antiproliferative agent to injured vasculature. Approximately 60-nm lipid-polymeric NPs were surface functionalized with collagen IV-targeting peptides and loaded with paclitaxel. In safety studies, the targeted NPs showed no signs of toxicity and a ≥3.5-fold improved maximum tolerated dose versus paclitaxel. In efficacy studies using a rat carotid injury model, paclitaxel (0.3 mg/kg or 1 mg/kg) was i.v. administered postprocedure on days 0 and 5. The targeted NP group resulted in lower neointima-to-media (N/M) scores at 2 wk versus control groups of saline, paclitaxel, or nontargeted NPs. Compared with sham-injury groups, an ∼50% reduction in arterial stenosis was observed with targeted NP treatment. The combination of improved tolerability, sustained release, and vascular targeting could potentially provide a safe and efficacious option in the management of CAD.

Structure of anthrax edema factor-calmodulin-adenosine 5'-(alpha,beta-methylene)-triphosphate complex reveals an alternative mode of ATP binding to the catalytic site.

Anthrax edema factor (EF) is a key virulence factor secreted by Bacillus anthracis. Here, we report a structure, at 3.0 A resolution, of the catalytic domain of EF (EF3) in complex with calmodulin (CaM) and adenosine 5'-(alpha,beta-methylene)-triphosphate (AMPCPP). Although the binding of the triphosphate of AMPCPP to EF3 can be superimposed on that of previously determined 3'deoxy-ATP (3'dATP) and 2'deoxy 3' anthraniloyl-ATP (2'd3' ANT-ATP) in EF3-CaM, the ribose and the adenine rings of AMPCPP are rotated approximately 105 and 180 degrees, respectively, relative to those of 3'dATP and 2'd3'ANT-ATP. Based on this model, K382 and F586 should play key roles in the recognition of adenine. However, mutations of these residues to alanine either separately or together cause only modest changes in Michaelis-Menten constants and IC50 values of AMPCPP and cAMP. Therefore, this alternate binding mode of the adenosine of AMPCPP binds to EF likely playing only a minor role in ATP binding and in catalysis.