Biphenyls as potent vitronectin receptor antagonists. Part 3: Squaric acid amides

Vitronectin receptor (alpha(V)beta(3)) antagonists have been implicated as a possible new treatment of restenosis following balloon angioplasty. In this work we investigate a series of novel arginine mimetic scaffolds leading to new insight of the alpha(V)beta(3)/ligand interaction. Squaric acid amide 10 is a subnanomolar alpha(V)beta(3) antagonist with improved potency on human smooth muscle cell migration.     

Genomic and experimental evidence for multiple metabolic functions in the RidA/YjgF/YER057c/UK114 (Rid) protein family

Background

It is now recognized that enzymatic or chemical side-reactions can convert normal metabolites to useless or toxic ones and that a suite of enzymes exists to mitigate such metabolite damage. Examples are the reactive imine/enamine intermediates produced by threonine dehydratase, which damage the pyridoxal 5''-phosphate cofactor of various enzymes causing inactivation. This damage is pre-empted by RidA proteins, which hydrolyze the imines before they do harm. RidA proteins belong to the YjgF/YER057c/UK114 family (here renamed the Rid family). Most other members of this diverse and ubiquitous family lack defined functions.

Results

Phylogenetic analysis divided the Rid family into a widely distributed, apparently archetypal RidA subfamily and seven other subfamilies (Rid1 to Rid7) that are largely confined to bacteria and often co-occur in the same organism with RidA and each other. The Rid1 to Rid3 subfamilies, but not the Rid4 to Rid7 subfamilies, have a conserved arginine residue that, in RidA proteins, is essential for imine-hydrolyzing activity. Analysis of the chromosomal context of bacterial RidA genes revealed clustering with genes for threonine dehydratase and other pyridoxal 5''-phosphate-dependent enzymes, which fits with the known RidA imine hydrolase activity. Clustering was also evident between Rid family genes and genes specifying FAD-dependent amine oxidases or enzymes of carbamoyl phosphate metabolism. Biochemical assays showed that Salmonella enterica RidA and Rid2, but not Rid7, can hydrolyze imines generated by amino acid oxidase. Genetic tests indicated that carbamoyl phosphate overproduction is toxic to S. enterica cells lacking RidA, and metabolomic profiling of Rid knockout strains showed ten-fold accumulation of the carbamoyl phosphate-related metabolite dihydroorotate.

Conclusions

Like the archetypal RidA subfamily, the Rid2, and probably the Rid1 and Rid3 subfamilies, have imine-hydrolyzing activity and can pre-empt damage from imines formed by amine oxidases as well as by pyridoxal 5''-phosphate enzymes. The RidA subfamily has an additional damage pre-emption role in carbamoyl phosphate metabolism that has yet to be biochemically defined. Finally, the Rid4 to Rid7 subfamilies appear not to hydrolyze imines and thus remain mysterious.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1584-3) contains supplementary material, which is available to authorized users.     

Thyroid hormones induce unique and potentially beneficial changes in cardiac myocyte shape in hypertensive rats near heart failure

We examined the effects of thyroid hormones (THs) on left ventricular (LV) function and myocyte remodeling in rats with spontaneously hypertensive heart failure (SHHF). SHHF rats were treated with three different TH doses from 20-21 mo of age. In terminal experiments, LV function (as determined by echocardiography and catheterization) and isolated myocyte shape were examined in SHHF rat groups and age-matched Wistar-Furth control animals. Compared with Wistar-Furth rats, the ratio of alpha- to beta-myosin was reduced in untreated SHHF rats. The alpha-to-beta-myosin ratio increased in all TH groups, which suggests a reversal of the fetal gene program. Low-dose TH produced no changes in LV myocyte size or function, but high-dose TH produced signs of hyperthyroidism (e.g., increased heart weight, tachycardia). The chamber diameter-to-wall thickness ratio declined with increasing dose due to reduced chamber diameter and increased wall thickness. This resulted in a 38% reduction in LV systolic wall stress in the middle- and high-dose groups despite sustained hypertension. Isolated myocyte data indicated that chamber remodeling and reduced wall stress were due to a unique alteration in myocyte transverse shape (e.g., reduced major diameter and increased minor diameter). Based on our present understanding of ventricular remodeling and wall stress, we believe these changes are likely beneficial. Results suggest that TH may be an important regulator of myocyte transverse shape in heart disease.     

MT1-MMP-dependent neovessel formation within the confines of the three-dimensional extracellular matrix

During angiogenesis, endothelial cells initiate a tissue-invasive program within an interstitial matrix comprised largely of type I collagen. Extracellular matrix-degradative enzymes, including the matrix metalloproteinases (MMPs) MMP-2 and MMP-9, are thought to play key roles in angiogenesis by binding to docking sites on the cell surface after activation by plasmin- and/or membrane-type (MT) 1-MMP-dependent processes. To identify proteinases critical to neovessel formation, an ex vivo model of angiogenesis has been established wherein tissue explants from gene-targeted mice are embedded within a three-dimensional, type I collagen matrix. Unexpectedly, neither MMP-2, MMP-9, their cognate cell-surface receptors (i.e., beta3 integrin and CD44), nor plasminogen are essential for collagenolytic activity, endothelial cell invasion, or neovessel formation. Instead, the membrane-anchored MMP, MT1-MMP, confers endothelial cells with the ability to express invasive and tubulogenic activity in a collagen-rich milieu, in vitro or in vivo, where it plays an indispensable role in driving neovessel formation.     

Immunological thresholds in neurological gene therapy: highly efficient elimination of transduced cells might be related to the specific formation of immunological synapses between T cells and virus-infected brain cells

First-generation adenovirus can be engineered with powerful promoters to drive expression of therapeutic transgenes. Numerous clinical trials for glioblastoma multiforme using first generation adenoviral vectors have either been performed or are ongoing, including an ongoing, Phase III, multicenter trial in Europe and Israel (Ark Therapeutics, Inc.). Although in the absence of anti-adenovirus immune responses expression in the brain lasts 6-18 months, systemic infection with adenovirus induces immune responses that inhibit dramatically therapeutic transgene expression from first generation adenoviral vectors, thus, potentially compromising therapeutic efficacy. Here, we show evidence of an immunization threshold for the dose that generates an immune response strong enough to eliminate transgene expression from the CNS. For the systemic immunization to eliminate transgene expression from the brain, > or = 1 x 10(7) infectious units (iu) of adenovirus need to be used as immunogen. Furthermore, this immune response eliminates >90% of transgene expression from 1 x 10(7)-1 x 10(3) iu of vector injected into the striatum 60 days earlier. Importantly, elimination of transgene expression is independent of the nature of the promoter that drives transgene expression and is accompanied by brain infiltration of CD8(+) T cells and macrophages. In conclusion, once the threshold for systemic immunization (i.e. 1 x 10(7) iu) is crossed, the immune response eliminates transgene expression by >90% even from brains that receive as little as 1000 iu of adenoviral vectors, independently of the type of promoter that drives expression.     

Influence of Ca, humic acid and pH on lead accumulation and toxicity in the fathead minnow during prolonged water-borne lead exposure

The present study examines the influence of Ca2+ as (CaSO4), dissolved organic carbon (DOC) and pH on chronic water-borne lead (Pb) toxicity to the larval fathead minnow (Pimephales promelas) under flow-through conditions. The 30 day LC50 for low hardness basic test water (19 mg CaCO3 L(-1)) was 39 (range: 27-51) microg dissolved Pb L(-1) and was greatly increased by increasing concentrations of CaSO4 and DOC to as much as 1903 (range: 1812-1992) mug dissolved Pb L(-1). Both reduced and increased pH (6.7 and 8.1, respectively) compared to control pH of 7.4 appeared to increase Pb toxicity substantially. Whole body Pb accumulation did not reflect water chemistry and thus exhibited no correlation with Pb induced mortality. One possible explanation for this lack of correlation is that mortality occurred predominantly during the first 4-6 days of exposure, whereas Pb accumulation was determined in surviving fish at the end of 30 days of exposure. Chronic Pb exposure resulted in a general iono-regulatory disturbance affecting K+, Na+ and Ca2+ homeostasis. However, recovery of Na+ and K+ levels and reversal of effects on Ca2+ homeostasis during continued exposure strongly suggest fathead minnow can acclimate to Pb. The gills accumulate the highest Pb concentrations during chronic exposure but the skeleton contains the largest mass of Pb by contributing up to approximately 80% of whole body Pb. In conclusion, water chemistry characteristics like Ca2+ and DOC should be considered for chronic water quality criteria.     

Development of tetravalent IgG1 dual targeting IGF-1R-EGFR antibodies with potent tumor inhibition

In this study we present novel bispecific antibodies that simultaneously target the insulin-like growth factor receptor type I (IGF-1R) and epidermal growth factor receptor (EGFR). For this purpose disulfide stabilized scFv domains of the EGFR/ADCC antibody GA201 were fused via serine-glycine connectors to the C-terminus of the heavy (XGFR2) or light chain (XGFR4), or the N-termini of the light (XGFR5) or heavy chain (XGFR3) of the IGF-1R antibody R1507 as parental IgG1 antibody. The resulting bispecific IGF-1R-EGFR antibodies XGFR2, XGFR3 and XGFR4 were successfully generated with yields and stability comparable to conventional IgG1 antibodies. They effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation of H322M and H460M2 tumor cells, induced strong down-modulation of IGF-1R as well as enhanced EGFR down-modulation compared to the parental EGFR antibody GA201 and were ADCC competent. The bispecific XGFR derivatives showed a strong format dependent influence of N- or C-terminal heavy and light chain scFv attachment on ADCC activity and an increase in receptor downregulation over the parental combination in vitro. XGFR2 and XGFR4 were selected for in vivo evaluation and showed potent anti-tumoral efficacy comparable to the combination of monospecific IGF-1R and EGFR antibodies in subcutaneous BxPC3 and H322M xenograft models. In summary, we have managed to overcome issues of stability and productivity of bispecific antibodies, discovered important antibody fusion protein design related differences on ADCC activity and receptor downmodulation and show that IGF-1R-EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components de-regulated in multiple cancer types, with the ultimate goal to avoid the formation of resistance to therapy.