Molecular dynamics simulations of CXCL-8 and its interactions with a receptor peptide, heparin fragments, and sulfated linked cyclitols

CXCL-8 (Interleukin 8) is a CXC chemokine with a central role in the human immune response. We have undertaken extensive in silico analyses to elucidate the interactions of CXCL-8 with its various binding partners, which are crucial for its biological function. Sequence and structure analyses showed that residues in the thirdq β-sheet and basic residues in the heparin binding site are highly variable, while residues in the second β-sheet are highly conserved. Molecular dynamics simulations in aqueous solution of dimeric CXCL-8 have been performed with starting geometries from both X-ray and NMR structures showed shearing movements between the two antiparallel C-terminal helices. Dynamic conservation analyses of these simulations agreed with experimental data indicating that structural differences between the two structures at quaternary level arise from changes in the secondary structure of the N-terminal loop, the 310-helix, the 30s, 40s, and 50s loops and the third β-sheet, resulting in a different interhelical separation. Nevertheless, the observation of these different states indicates that CXCL-8 has the potential to undergo conformational changes, and it seems likely that this feature is relevant to the mode of binding of glycosaminoglycan (GAG) mimetics such as cyclitols. Simulations of the receptor peptide fragment−CXCL-8 complex identified several specific interactions of the receptor peptide with CXCL-8 that could be exploited in the structure-based design of competitive peptides and nonpeptidic molecules targeting CXCL-8 for combating inflammatory diseases. Simulations of the CXCL-8 dimer complexed with a 24-mer heparin fragment and of the CXCL-8−receptor peptide complex revealed that Arg60, Lys64, and Arg68 in the dimer bind to cyclitols in a horseshoe pattern, defining a region which is spatially distinct from the receptor binding site. There appears to be an optimum number of sulfates and an optimum length of alkyl spacers required for the interaction of cyclitol inhibitors with the dimeric form of CXCL-8. Calculation of the binding affinities of cyclitol inhibitors reflected satisfactorily the ranking of experimentally determined inhibitory potencies. The findings of these molecular modeling studies will help in the search for inhibitors which can modulate various CXCL-8 biological activities and serve as an excellent model system to study CXC-inhibitor interactions.

The PG500 series : novel heparan sulfate mimetics as potent angiogenesis and heparanase inhibitors for cancer therapy

Heparan sulfate mimetics, which we have called the PG500 series, have been developed to target the inhibition of both angiogenesis and heparanase activity. This series extends the technology underpinning PI-88, a mixture of highly sulfated oligosaccharides which reached Phase III clinical development for hepatocellular carcinoma. Advances in the chemistry of the PG500 series provide numerous advantages over PI-88. These new compounds are fully sulfated, single entity oligosaccharides attached to a lipophilic moiety, which have been optimized for drug development. The rational design of these compounds has led to vast improvements in potency compared to PI-88, based on in vitro angiogenesis assays and in vivo tumor models. Based on these and other data, PG545 has been selected as the lead clinical candidate for oncology and is currently undergoing formal preclinical development as a novel treatment for advanced cancer.

Coating of biomaterial scaffolds with the collagen-mimetic peptide GFOGER for bone defect repair

Healing large bone defects and non-unions remains a significant clinical problem. Current treatments, consisting of auto and allografts, are limited by donor supply and morbidity, insufficient bioactivity and risk of infection. Biotherapeutics, including cells, genes and proteins, represent promising alternative therapies, but these strategies are limited by technical roadblocks to biotherapeutic delivery, cell sourcing, high cost, and regulatory hurdles. In the present study, the collagen-mimetic peptide, GFOGER, was used to coat synthetic PCL scaffolds to promote bone formation in critically-sized segmental defects in rats. GFOGER is a synthetic triple helical peptide that binds to the [alpha]2[beta]1 integrin receptor involved in osteogenesis. GFOGER coatings passively adsorbed onto polymeric scaffolds, in the absence of exogenous cells or growth factors, significantly accelerated and increased bone formation in non-healing femoral defects compared to uncoated scaffolds and empty defects. Despite differences in bone volume, no differences in torsional strength were detected after 12 weeks, indicating that bone mass but not bone quality was improved in this model. This work demonstrates a simple, cell/growth factor-free strategy to promote bone formation in challenging, non-healing bone defects. This biomaterial coating strategy represents a cost-effective and facile approach, translatable into a robust clinical therapy for musculoskeletal applications.

Prandial subcutaneous injections of glucagon-like peptide-1 cause weight loss in obese human subjects

Recombinant glucagon-like peptide-1 (7–36)amide (rGLP-1) was recently shown to cause significant weight loss in type 2 diabetics when administered for 6 weeks as a continuous subcutaneous infusion. The mechanisms responsible for the weight loss are not clarified. In the present study, rGLP-1 was given for 5d by prandial subcutaneous injections (PSI) (76nmol 30min before meals, four times daily; a total of 302·4nmol/24h) or by continuous subcutaneous infusion (CSI) (12·7nmol/h; a total of 304·8nmol/24h). This was performed in nineteen healthy obese subjects (mean age 44·2 (sem 2·5) years; BMI 39·0 (sem 1·2)kg/m2) in a prospective randomised, double-blind, placebo-controlled, cross-over study. Compared with the placebo, rGLP-1 administered as PSI and by CSI generated a 15% reduction in mean food intake per meal (P=0·02) after 5d treatment. A weight loss of 0·55 (sem 0·2) kg (P<0·05) was registered after 5d with PSI of rGLP-1. Gastric emptying rate was reduced during both PSI (P<0·001) and CSI (P<0·05) treatment, but more rapidly and to a greater extent with PSI of rGLP-1. To conclude, a 5d treatment of rGLP-1 at high doses by PSI, but not CSI, promptly slowed gastric emptying as a probable mechanism of action of increased satiety, decreased hunger and, hence, reduced food intake with an ensuing weight loss.

The expanding roles of the ghrelin-gene derived peptide obestatin in health and disease

Obestatin is a 23 amino acid, ghrelin gene-derived peptide hormone produced in the stomach and a range of other tissues throughout the body. While it was initially reported that obestatin opposed the actions of ghrelin with regards to appetite and food intake, it is now clear that obestatin is not an endogenous ghrelin antagonist of ghrelin, but it is a multi-functional peptide hormone in its own right. In this review we will discuss the controversies associated with the discovery of obestatin and explore emerging central and peripheral roles of obestatin, roles in adipogenesis, pancreatic homeostasis and cancer.

A fragment of the LG3 peptide of endorepellin is present in the urine of physically active mining workers : a potential marker of physical activity

Biomarker analysis has been implemented in sports research in an attempt to monitor the effects of exertion and fatigue in athletes. This study proposed that while such biomarkers may be useful for monitoring injury risk in workers, proteomic approaches might also be utilised to identify novel exertion or injury markers. We found that urinary urea and cortisol levels were significantly elevated in mining workers following a 12 hour overnight shift. These levels failed to return to baseline over 24h in the more active maintenance crew compared to truck drivers (operators) suggesting a lack of recovery between shifts. Use of a SELDI-TOF MS approach to detect novel exertion or injury markers revealed a spectral feature which was associated with workers in both work categories who were engaged in higher levels of physical activity. This feature was identified as the LG3 peptide, a C-terminal fragment of the anti-angiogenic / anti-tumourigenic protein endorepellin. This finding suggests that urinary LG3 peptide may be a biomarker of physical activity. It is also possible that the activity mediated release of LG3 / endorepellin into the circulation may represent a biological mechanism for the known inverse association between physical activity and cancer risk / survival.

Expression Pattern of the Alpha-Kafirin Promoter Coupled with a Signal Peptide from Sorghum bicolor L. Moench

Regulatory sequences with endosperm specificity are essential for foreign gene expression in the desired tissue for both grain quality improvement and molecular pharming. In this study, promoters of seed storage α-kafirin genes coupled with signal sequence (ss) were isolated from Sorghum bicolor L. Moench genomic DNA by PCR. The α-kafirin promoter (α-kaf) contains endosperm specificity-determining motifs, prolamin-box, the O2-box 1, CATC, and TATA boxes required for α-kafirin gene expression in sorghum seeds. The constructs pMB-Ubi-gfp and pMB-kaf-gfp were microprojectile bombarded into various sorghum and sweet corn explants. GFP expression was detected on all explants using the Ubi promoter but only in seeds for the α-kaf promoter. This shows that the α-kaf promoter isolated was functional and demonstrated seed-specific GFP expression. The constructs pMB-Ubi-ss-gfp and pMB-kaf-ss-gfp were also bombarded into the same explants. Detection of GFP expression showed that the signal peptide (SP)::GFP fusion can assemble and fold properly, preserving the fluorescent properties of GFP.