Circulatory therapeutics: use of antihypertensive agents and their effects on the vasculature

This review addresses the use of the different antihypertensive agents currently available and some in development, and their effects on the vasculature. The different classes of agents used in the treatment of hypertension, and the results of recent large clinical trials, dosing protocols and adverse effects are first briefly summarized. The consequences on blood vessels of the use of antihypertensive drugs and the differential effects on the biology of large and small arteries resulting in modulation of vascular remodelling and dysfunction in hypertensive patients are then described. Large elastic conduit arteries exhibit outward hypertrophic remodelling and increased stiffness, which contributes to raise systolic blood pressure and afterload on the heart. Small resistance arteries undergo eutrophic or hypertrophic inward remodelling, and impair tissue perfusion. By these mechanisms both large and small arteries may contribute to trigger cardiovascular events. Some antihypertensive agents correct these changes, which could contribute to improved outcome. The mechanisms that at the level of the vascular wall lead to remodelling and can be beneficially affected by antihypertensive agents will also be addressed. These include vasoconstriction, growth and inflammation. The molecular pathways contributing to growth and inflammation will be summarily described. Further identification of these signalling pathways should allow identification of novel targets leading to development of new and improved medications for the treatment of hypertension and cardiovascular disease.     

β‐amino acid substitution to investigate the recognition of angiotensin II (AngII) by angiotensin converting enzyme 2 (ACE2)

In spite of the important role of angiotensin converting enzyme 2 (ACE2) in the cardiovascular system, little is known about the substrate structural requirements of the AngII–ACE2 interaction. Here we investigate how changes in angiotensin II (AngII) structure affect binding and cleavage by ACE2. A series of C3 β‐amino acid AngII analogs were generated and their secondary structure, ACE2 inhibition, and proteolytic stability assessed by circular dichroism (CD), quenched fluorescence substrate (QFS) assay, and LC‐MS analysis, respectively. The β‐amino acid‐substituted AngII analogs showed differences in secondary structure, ACE2 binding and proteolytic stability. In particular, three different subsets of structure‐activity profiles were observed corresponding to substitutions in the N‐terminus, the central region and the C‐terminal region of AngII. The results show that β‐substitution can dramatically alter the structure of AngII and changes in structure correlated with ACE2 inhibition and/or substrate cleavage. β‐amino acid substitution in the N‐terminal region of AngII caused little change in structure or substrate cleavage, while substitution in the central region of AngII lead to increased β‐turn structure and enhanced substrate cleavage. β‐amino acid substitution in the C‐terminal region significantly diminished both secondary structure and proteolytic processing by ACE2. The β‐AngII analogs with enhanced or decreased proteolytic stability have potential application for therapeutic intervention in cardiovascular disease. Copyright © 2010 John Wiley & Sons, Ltd.     

TGF‐β1 rs1982073 polymorphism contributes to radiation pneumonitis in lung cancer patients: a meta‐analysis

Transforming growth factor beta 1(TGF‐β1) polymorphism was associated with radiation pneumonitis (RP) susceptibility, but their results have been inconsistent. The PubMed and CNKI were searched for case‐control studies published up to Januray 01, 2016 was Data were extracted and pooled odds ratios (OR) with 95% confidence intervals (CI) were calculated. In this meta‐analysis, we assessed eight publications involving 368 radiation pneumonitis cases and 855 controls of the association between TGF‐β1 T869C (rs1982073) and G915C (rs1800471) polymorphism and RP susceptibility. Our analysis suggested that TGF‐β1 T869C rs1982073 polymorphism was associated with lower RP risk for CT combined CC versus TT model (OR = 0.58, 95% CI = 0.43–0.77). However, for the G915C rs1800471 polymorphism, no association was found between the polymorphism and the susceptibility to RP in GC combined CC versus GG model (OR = 0.82, 95% CI = 0.50–1.35). These results from the meta‐analysis suggest that T869C rs1982073 polymorphism of TGF‐β1 may be associated with RP risk, and there may be no association between G915C polymorphism and RP risk.     

The variant at TGFBRAP1 is significantly associated with type 2 diabetes mellitus and affects diabetes‐related miRNA expression

While the transforming growth factor‐β1 (TGF‐β1) regulates the growth and proliferation of pancreatic β‐cells, its receptors trigger the activation of Smad network and subsequently induce the insulin resistance. A case‐control was conducted to evaluate the associations of the polymorphisms of TGF‐β1 receptor‐associated protein 1 (TGFBRAP1) and TGF‐β1 receptor 2 (TGFBR2) with type 2 diabetes mellitus (T2DM), and its genetic effects on diabetes‐related miRNA expression. miRNA microarray chip was used to screen T2DM‐related miRNA and 15 differential expressed miRNAs were further validated in 75 T2DM and 75 normal glucose tolerance (NGT). The variation of rs2241797 (T/C) at TGFBRAP1 showed significant association with T2DM in case‐control study, and the OR (95% CI) of dominant model for cumulative effects was 1.204 (1.060‐1.370), Bonferroni corrected P < 0.05. Significant differences in the fast glucose and HOMA‐β indices were observed amongst the genotypes of rs2241797. The expression of has‐miR‐30b‐5p and has‐miR‐93‐5p was linearly increased across TT, TC, and CC genotypes of rs2241797 in NGT, P_trend values were 0.024 and 0.016, respectively. Our findings suggest that genetic polymorphisms of TGFBRAP1 may contribute to the genetic susceptibility of T2DM by mediating diabetes‐related miRNA expression.     

Biocatalytic Approach for the Synthesis of Enantiopure Acebutolol as a β1‐Selective Blocker

A new chemoenzymatic route is reported to synthesize acebutolol, a selective β₁ adrenergic receptor blocking agent in enantiopure (R and S) forms. The enzymatic kinetic resolution strategy was used to synthesize enantiopure intermediates (R)‐ and (S)‐N‐(3‐acetyl‐4‐(3‐chloro‐2‐hydroxypropoxy)phenyl)butyramide from the corresponding racemic alcohols. The results showed that out of eleven commercially available lipase preparations, two enzyme preparations (Lipase A, Candida antarctica, CLEA [CAL CLEA] and Candida rugosa lipase, 62316 [CRL 62316]) act in enantioselective manner. Under optimized conditions the enantiomeric excess of both (R)‐ and (S)‐N‐(3‐acetyl‐4‐(3‐chloro‐2‐hydroxypropoxy)phenyl)butyramide were 99.9 and 96.8%, respectively. N‐alkylation of both the (R) and (S) intermediates with isopropylamine gave enantiomerically pure (R and S)‐ acebutolol with a yield 68 and 72%, respectively. This study suggests a high yielding, easy and environmentally green approach to synthesize enantiopure acebutolol. Chirality 27:382–391, 2015. © 2015 Wiley Periodicals, Inc.     

KCNQ1 rs2237892 C→T gene polymorphism and type 2 diabetes mellitus in the Asian population: a meta‐analysis of 15,736 patients

The KCNQ1 rs2237892 C→T gene polymorphism is reportedly associated with T2DM susceptibility, but various studies show conflicting results. To explore this association in the Asian population, a meta‐analysis of 15,736 patients from 10 individual studies was performed. The pooled odds ratios (ORs) and their 95% confidence intervals (CIs) were evaluated using random‐effect or fixed‐effect models. A significant relationship between the KCNQ1 rs2237892 C→T gene polymorphism and T2DM was observed in the Asian population under the allelic (OR, 1.350; 95% CI, 1.240–1.480; P < 0.00001), recessive (OR: 0.650; 95% CI: 0.570–0.730; P < 0.00001), dominant (OR: 1.450; 95% CI: 1.286–1.634; P < 0.00001), and additive (OR: 1.346; 95% CI: 1.275–1.422; P < 0.00001) genetic models. In the subgroup analysis by race, a significant association was found in Chinese, Korean and Malaysia population, but not in Indian population. KCNQ1 rs2237892 C→T gene polymorphism was found to be significantly associated with increased T2DM risk in the Asian population, except Indian population. The C allele of the KCNQ1 rs2237892 C→T gene polymorphism may confer susceptibility to T2DM.     

Insight into a strategy for attenuating AmpC‐mediated β‐lactam resistance: Structural basis for selective inhibition of the glycoside hydrolase NagZ

NagZ is an exo‐N‐acetyl‐β‐glucosaminidase, found within Gram‐negative bacteria, that acts in the peptidoglycan recycling pathway to cleave N‐acetylglucosamine residues off peptidoglycan fragments. This activity is required for resistance to cephalosporins mediated by inducible AmpC β‐lactamase. NagZ uses a catalytic mechanism involving a covalent glycosyl enzyme intermediate, unlike that of the human exo‐N‐acetyl‐β‐glucosaminidases: O‐GlcNAcase and the β‐hexosaminidase isoenzymes. These latter enzymes, which remove GlcNAc from glycoconjugates, use a neighboring‐group catalytic mechanism that proceeds through an oxazoline intermediate. Exploiting these mechanistic differences we previously developed 2‐N‐acyl derivatives of O‐(2‐acetamido‐2‐deoxy‐D ‐glucopyranosylidene)amino‐N‐phenylcarbamate (PUGNAc), which selectively inhibits NagZ over the functionally related human enzymes and attenuate antibiotic resistance in Gram‐negatives that harbor inducible AmpC. To understand the structural basis for the selectivity of these inhibitors for NagZ, we have determined its crystallographic structure in complex with N‐valeryl‐PUGNAc, the most selective known inhibitor of NagZ over both the human β‐hexosaminidases and O‐GlcNAcase. The selectivity stems from the five‐carbon acyl chain of N‐valeryl‐PUGNAc, which we found ordered within the enzyme active site. In contrast, a structure determination of a human O‐GlcNAcase homologue bound to a related inhibitor N‐butyryl‐PUGNAc, which bears a four‐carbon chain and is selective for both NagZ and O‐GlcNAcase over the human β‐hexosamnidases, reveals that this inhibitor induces several conformational changes in the active site of this O‐GlcNAcase homologue. A comparison of these complexes, and with the human β‐hexosaminidases, reveals how selectivity for NagZ can be engineered by altering the 2‐N‐acyl substituent of PUGNAc to develop inhibitors that repress AmpC mediated β‐lactam resistance.     

Functional analysis and purification of a Pen‐2 fusion protein for γ‐secretase structural studies

The 19‐transmembrane, multisubunit γ‐secretase complex generates the amyloid β‐peptide (Aβ) of Alzheimer's disease (AD) by an unusual intramembrane proteolysis of the β‐amyloid precursor protein. The complex, which similarly processes many other type 1 transmembrane substrates, is composed of presenilin, Aph1, nicastrin, and presenilin enhancer (Pen‐2), all of which are necessary for proper complex maturation and enzymatic activity. Obtaining a high‐resolution atomic structure of the intact complex would greatly aid the rational design of compounds to modulate activity but is a very difficult task. A complementary method is to generate structures for each individual subunit to allow one to build a model of the entire complex. Here, we describe a method by which recombinant human Pen‐2 can be purified from bacteria to > 95% purity at milligram quantities per liter, utilizing a maltose binding protein tag to both increase solubility and facilitate purification. Expressing the same construct in mammalian cells, we show that the large N‐terminal maltose binding protein tag on Pen‐2 still permits incorporation into the complex and subsequent presenilin‐1 endoproteolysis, nicastrin glycosylation and proteolytic activity. These new methods provide valuable tools to study the structure and function of Pen‐2 and the γ‐secretase complex.

     

Designing anti‐diabetic β‐cells microcapsules using polystyrenic sulfonate,polyallylamine, and a tertiary bile acid: Morphology,bioenergetics, and cytokine analysis

Purpose: Recently sodium alginate (SA)‐poly‐l‐ornithine (PLO) microcapsules containing pancreatic β‐cells that showed good morphology but low cell viability (<27%) was designed. In this study, two new polyelectrolytes, polystyrenic sulfonate (PSS; at 1%) and polyallylamine (PAA; at 2%) were incorporated into a microencapsulated‐formulation, with the aim of enhancing the physical properties of the microcapsules. Following incorporation, the structural characteristics and cell viability were investigated. The effects of the anti‐inflammatory bile acid, ursodeoxycholic acid (UDCA), on microcapsule morphology, size, and stability as well as β‐cell biological functionality was also examined. Methods: Microcapsules were prepared using PLO‐PSS‐PAA‐SA mixture and two types of microcapsules were produced: without UDCA (control) and with UDCA (test). Microcapsule morphology, stability, and size were examined. Cell count, microencapsulation efficiency, cell bioenergetics, and activity were also examined. Results: The new microcapsules showed good morphology but cell viability remained low (29% ± 3%). UDCA addition improved cell viability post‐microencapsulation (42 ± 5, P < 0.01), reduced swelling (P < 0.01), improved mechanical strength (P < 0.01), increased Zeta‐potential (P < 0.01), and improved stability. UDCA addition also increased insulin production (P < 0.01), bioenergetics (P < 0.01), and decreased β‐cell TNF‐α (P < 0.01), IFN‐gamma (P < 0.01), and IL‐6 (P < 0.01) secretions. Conclusions: Addition of 4% UDCA to a formulation system consisting of 1.8% SA, 1% PLO, 1% PSS, and 2% PAA enhanced cell viability post‐microencapsulation and resulted in a more stable formulation with enhanced encapsulated β‐cell metabolism, bioenergetics, and biological activity with reduced inflammation. This suggests potential application of UDCA, when combined with SA, PLO, PSS, and PAA, in β‐cell microencapsulation and diabetes treatment. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:501–509, 2016     

Conformational flexibility and loss of structural rigidity for a model hexapeptide,GRGDTP: 1H‐NMR and molecular dynamics studies

The NMR and molecular dynamics methods are used to study the conformations of a hexapeptide, GRGDTP, which has been shown to be accessible to various types of cell‐adhesion based cellular behaviors such as cell‐to‐matrix interactions, cell differentiation, immunogenicity development, gene expression, angiogenesis, metastasis, sex determination and gamete fusion. ¹H‐NMR results indicate the existence of weak 5→2 hydrogen bonded β‐turn type‐III. Molecular simulation studies using a mixed protocol of distance geometry, constrained minimization, restrained molecular dynamics followed by energy minimization resulted additional conformations that include about 64% of population of inverse γ‐turn (HB, 3→1) and about 35% population of γ‐turn (HB, 4→2). The inter‐proton distances observed in γ‐and inverse γ‐turns are also consistent with the NMR constraints. The variable internal hydrogen bonding due to γ‐turns initiated at Gly 1 and Arg 2 , and its tendency to inter‐convert between γ‐and inverse γ‐turn conformations imply that the peptide is flexible in nature. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 460–471, 2013.     

Quantitative analysis of the secretome of TGF‐β signaling‐deficient mammary fibroblasts

Transforming growth factor β (TGF‐β) is a master regulator of autocrine and paracrine signaling pathways between a tumor and its microenvironment. Decreased expression of TGF‐β type II receptor (TβRII) in stromal cells is associated with increased tumor metastasis and shorter patient survival. In this study, SILAC quantitative proteomics was used to identify differentially externalized proteins in the conditioned media from the mammary fibroblasts with or without intact TβRII. Over 1000 proteins were identified and their relative differential levels were quantified. Immunoassays were used to further validate identification and quantification of the proteomic results. Differential expression was detected for various extracellular proteins, including proteases and their inhibitors, growth factors, cytokines, and extracellular matrix proteins. CXCL10, a cytokine found to be up‐regulated in the TβRII knockout mammary fibroblasts, is shown to directly stimulate breast tumor cell proliferation and migration. Overall, this study revealed hundreds of specific extracellular protein changes modulated by deletion of TβRII in mammary fibroblasts, which may play important roles in the tumor microenvironment. These results warrant further investigation into the effects of inhibiting the TGF‐β signaling pathway in fibroblasts because systemic inhibition of TGF‐β signaling pathways is being considered as a potential cancer therapy.     

A new fluorescent chemosensor for cadmium(II) based on a pyrene‐appended piperidone derivative and its β‐cyclodextrin complex

We report, in this article, a piperidin‐4‐one derivative carrying pyrenyl fluorescent reporter groups which acts as a Cd²⁺ ion sensor. The compound is synthesized and characterized using IR and NMR spectral techniques. The compound forms an inclusion complex with β‐cyclodextrin. It selectively binds to Cd²⁺ ions in water and aqueous β‐cyclodextrin media. The stoichiometry of the host–guest complex of the compound with β‐cyclodextrin is 1:2. The ligand–metal ion binding stoichiometry is 1:1 both in water and in β‐cyclodextrin. The linear concentration range of detection of the metal ion is reported. Cyclodextrin complex formation does not affect the metal ion selectivity of the compound.     

Developing bifunctional β‐lactamase molecules with built‐in target‐recognizing module for prodrug therapy: identification of Enterobacter Cloacae P99 cephalosporinase loops suitable for randomization and phage‐display selection

This study was focused on developing catalytically active β‐lactamase enzyme molecules that have target‐recognizing sites built within their scaffold. Using phage‐display approach, nine libraries were constructed by inserting the randomized linear or cysteine‐constrained heptapeptides in the five different loops on the outer surface of P99 β‐lactamase molecule. The pIII signal peptide of Sec‐pathway was employed for a periplasmic translocation of the β‐lactamase fusion protein, which we found more efficient than the DsbA signal peptide of SRP‐pathway. The randomized heptapeptide loops replaced native amino acids between positions ³⁴Y‐³⁷K, ²³⁸M‐²⁴⁶A, ²⁷⁵N‐²⁸⁰A, ³⁰⁵A‐³¹¹S, or ³²⁹I‐³³⁴I of the P99 β‐lactamase molecules for generating the loop‐1 to ‐5 libraries, respectively. The diversity of each loop library was judged by counting the primary and β‐lactamase‐active clones. The linear peptide inserts in the loop‐2 library showed the maximum number of the β‐lactamase‐active clones, followed by the loop‐5, loop‐3, and loop‐4. The insertion of the cysteine‐constrained loops exhibited a dramatic loss of the enzyme‐active β‐lactamase clones. The complexity of the loop‐2 linear library, as determined by the frequency and diversity of amino acid distributions in the randomized region, appears consistent with the standards of other types of phage display library systems. The selection of the loop‐2 linear library on streptavidin protein as a test target identified several β‐lactamase clones that specifically bound to streptavidin. In conclusion, this study identified the suitability of the loop‐2 of P99 β‐lactamase for constructing a phage‐display library of the β‐lactamase enzyme‐active molecules that can be selected against a target. This is an enabling step in our long‐term goal of developing bifunctional β‐lactamase molecules against cancer‐specific targets for enzyme prodrug therapy of cancer. Copyright © 2009 John Wiley & Sons, Ltd.     

Crystal structure analysis reveals a spring‐loaded latch as molecular mechanism for GDF‐5–type I receptor specificity

Dysregulation of growth and differentiation factor 5 (GDF‐5) signalling, a member of the TGF‐β superfamily, is strongly linked to skeletal malformation. GDF‐5‐mediated signal transduction involves both BMP type I receptors, BMPR‐IA and BMPR‐IB. However, mutations in either GDF‐5 or BMPR‐IB lead to similar phenotypes, indicating that in chondrogenesis GDF‐5 signalling seems to be exclusively mediated through BMPR‐IB. Here, we present structural insights into the GDF‐5:BMPR‐IB complex revealing how binding specificity for BMPR‐IB is generated on a molecular level. In BMPR‐IB, a loop within the ligand‐binding epitope functions similar to a latch allowing high‐affinity binding of GDF‐5. In BMPR‐IA, this latch is in a closed conformation leading to steric repulsion. The new structural data now provide also a molecular basis of how phenotypically relevant missense mutations in GDF‐5 might impair receptor binding and activation.