XTEN, unstructured biodegradable proteins, have been used to extend the in vivo half-life of genetically fused therapeutic proteins and peptides. To expand the applications of XTEN technology to half-life extension of other classes of molecules, XTEN protein polymers and methods for chemical XTENylation were developed. Two XTEN precursors were engineered to contain enzymatically removable purification tags. The proteins were readily expressed in bacteria and purified to homogeneity by chromatography techniques. As proof-of-principle, GLP2-2G peptide was chemically conjugated to each of the two XTEN protein polymers using maleimide-thiol chemistry. The monodisperse nature of XTEN protein polymer enabled reaction monitoring as well as the detection of peptide modifications in the conjugated state using reverse phase-high performance liquid chromatography (RP-HPLC) and electrospray ionization mass spectrometry. The resulting GLP2-2G-XTEN conjugates were purified by preparative RP-HPLC to homogeneity. In comparison with recombinantly fused GLP2-2G-XTEN, chemically conjugated GLP2-2G-XTEN molecules exhibited comparable in vitro activity, in vitro plasma stability and pharmacokinetics in rats. These data suggest that chemical XTENylation could effectively extend the half-life of a wide spectrum of biologically active molecules, therefore broadening its applicability.
XTENs are unstructured, nonrepetitive
protein polymers designed
to prolong the in vivo half-life of pharmaceuticals by introducing
a bulking effect similar to that of poly(ethylene glycol). While XTEN
can be expressed as a recombinant fusion protein with bioactive proteins
and peptides, therapeutic molecules of interest can also be chemically
conjugated to XTEN. Such an approach permits precise control over
the positioning, spacing, and valency of bioactive moieties along
the length of XTEN. We have demonstrated the attachment of T-20, an
anti-retroviral peptide indicated for the treatment of HIV-1 patients
with multidrug resistance, to XTEN. By reacting maleimide-functionalized
T-20 with cysteine-containing XTENs and varying the number and positioning
of cysteines in the XTENs, a library of different peptide–polymer
combinations were produced. The T-20-XTEN conjugates were tested using
an in vitro antiviral assay and were found to be effective in inhibiting
HIV-1 entry and preventing cell death, with the copy number and spacing
of the T-20 peptides influencing antiviral activity. The peptide–XTEN
conjugates were also discovered to have enhanced solubilities in comparison
with the native T-20 peptide. The pharmacokinetic profile of the most
active T-20-XTEN conjugate was measured in rats, and it was found
to exhibit an elimination half-life of 55.7 ± 17.7 h, almost
20 times longer than the reported half-life for T-20 dosed in rats.
As the conjugation of T-20 to XTEN greatly improved the in vivo half-life
and solubility of the peptide, the XTEN platform has been demonstrated
to be a versatile tool for improving the properties of drugs and enabling
the development of a class of next-generation therapeutics.
Previous studies have shown that the RAS (renin-angiotensin system) might participate in airway remodelling in asthma. As a main component of the RAS, Ang-(1-7) [angiotensin-(1-7)] has been reported in few studies regarding its protective effect on asthma. However, the functional roles and relevant signalling pathways of Ang-(1-7) have not been well illustrated. In the present study, we analysed the effect of Ang-(1-7) on AngII (angiotensin II)-induced HLF (human lung fibroblast)-MF (myofibroblast) transition by detecting Col-I (collagen type I), TGF-β1 (transforming growth factor-β1) and α-SMA (α-smooth muscle actin) expression. We explored further the possible signalling pathways involved in HLF-MF transition. Our results showed that Ang-(1-7) could down-regulate the expression of Col-I, α-SMA and TGF-β1/Smad2/3 (all P<0.05). A significant decrease was found in phosphorylation of PI3K (phosphoinositide 3-kinase), Akt, p38-MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase) signalling pathways during HLF-MF transition (all P<0.05). Our data suggests that Ang-(1-7) decreases the expression of Col-I via TGF-β1/Smad2/3 and subsequently inhibits HLF-MF transition.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.