Serpin†A1 C-Terminal Peptides as Collagen Turnover Modulators.

The modulation of collagen turnover can be a relevant pharmacological target in the context of treating either pathological or pathophysiological conditions, such as collagen-related diseases and skin aging. Our recent work has focused on the search for short-chain peptides as lead compounds for further development of compounds that enhance the production of type I collagen. In this study we selected and synthesized overlapping peptides of the C-terminal portion of serpin A1 (residues 393-418), the impact of which on collagen production has been reported previously, in order to identify shorter and still active fragments and to provide insight on the mechanisms involved. The biological activity of each fragment was evaluated with cultured normal human dermal fibroblasts, and changes in the amounts of collagen were monitored in collected culture media by a sandwich ELISA technique developed in house. Interestingly, we identified a decapeptide, termed SA1-III (Ac-MGKVVNPTQK-NH2 ), as a promising candidate for our purposes; it is able to induce a significant increase in type I collagen levels in the culture medium of treated cells at micromolar concentrations.

[The current progress in the development of HIV-1 fusion inhibitors].

HIV-1 fusion inhibitors are a new class of anti-HIV compounds, which block the entry of HIV into target cells through preventing the fusion between viral and cell plasma membrane and thus interrupt the initial steps of viral replication. T-20 (enfuvirtide), which has been clinically approved as the first fusion inhibitor of HIV-1 by U.S. FDA in 2003, can suppress replication of HIV variants with multi-drug resistance to reverse transcriptase and protease inhibitors. Peptides and small molecules display potent anti-HIV fusion activities by targeting gp41 thus inhibit its fusogenic function. In recent years, with the development of studies on the molecular mechanism of HIV membrane fusion process and the function of gp41, many new fusion inhibitors are found and some have been in advanced clinical trials. This review discusses recent progress in the development of HIV-1 fusion inhibitors targeting the gp41.

Different conformational changes within the F-helix occur during serpin folding, polymerization, and proteinase inhibition.

The intrinsic metastability of the serpin native state is the thermodynamic driving force for both proteinase inhibition and the formation of inactive polymers. A number of mechanisms has been proposed to explain how both these conformational changes are achieved. However, one aspect that has received little attention is the movement of the F-helix, which physically impedes both these events. We have applied a protein engineering approach to investigate the conformational changes of this helix during proteinase inhibition, serpin folding, and polymerization. We systematically mutated two highly conserved hydrophobic residues on the F-helix, V161 and I157, and in addition, removed a hydrogen bond between D149 and the first turn of the helix. Our data demonstrate that while all three interactions are important for the stability and folding of the molecule, their contribution during inhibition and polymerization differ. The presence of I157 is crucial to all conformational changes as its loss results in inactivation of the serpin and rapid polymerization. The replacement of D149 does not affect activity but significantly increases the polymerization rate. The interactions formed by V161 play an important role only in maintaining the native conformation. Taken together, these data suggest that the F-helix undergoes a reversible conformational change in both its N- and C-termini during proteinase inhibition only the C-terminus undergoes changes during polymerization, but there is a global change required for folding.

Evaluation of the rapid plasma elimination of recombinant alpha 1-proteinase inhibitor: synthesis of polyethylene glycol conjugates with improved therapeutic potential.

Plasma-derived alpha 1-proteinase inhibitor (alpha 1PlI) is used for replacement therapy in patients with emphysema who have a deficiency of the protein. Future therapy with alpha 1PI with reactive site mutants of this inhibitor will probably require the use of recombinant-derived alpha 1PI (r alpha 1PI). However, the pharmacologic efficacy of r alpha 1PI in humans has been hindered, since r alpha 1PI is rapidly cleared from the circulation of experimental animals. Our studies of the metabolism of r alpha 1PI in mice demonstrate that the rapid clearance of r alpha 1PI is due to renal filtration. However, after preventing renal filtration by ligation of the renal arteries, we find that r alpha 1PI is metabolized in a manner similar to alpha 1PI. Conjugation of r alpha 1PI to polyethylene glycol of Mr approximately 4000 (PEG-4) greatly slows the clearance of r alpha 1PI. The PEG-4 r alpha 1PI conjugate is metabolized in a manner similar to alpha 1PI and has similar kinetic properties before and after oxidation with N-chlorosuccinimide. Moreover, proteinase complexes of PEG-4-r alpha 1PI are catabolized by the same hepatocyte receptor that binds alpha 1PI-proteinase complexes. The results of these studies lay the groundwork for the synthesis of pharmacologically effective r alpha 1PI derivatives with acceptable plasma retention times.