Preexposure prophylaxis with albumin-conjugated C34 peptide HIV-1 fusion inhibitor in SCID-hu Thy/Liv mice.

PC-1505 is a C34 peptide derived from the heptad repeat 2 region of HIV-1 gp41 conjugated to human serum albumin for sustained in vivo activity. One single preexposure dose of PC-1505 reduced viral RNA in HIV-1-infected SCID-hu Thy/Liv mice by 3.3 log10 and protected T cells from virus-mediated depletion. In contrast, a single preexposure dose of Truvada reduced viral RNA by only 0.8 log10 and was substantially less effective in preventing T cell depletion.

Albumin-conjugated C34 peptide HIV-1 fusion inhibitor: equipotent to C34 and T-20 in vitro with sustained activity in SCID-hu Thy/Liv mice.

Entry inhibitors of human immunodeficiency virus, type 1 (HIV-1) have been the focus of much recent research. C34, a potent fusion inhibitor derived from the HR2 region of gp41, was engineered into a 1:1 human serum albumin conjugate through stable covalent attachment of a maleimido-C34 analog onto cysteine 34 of albumin. This bioconjugate, PC-1505, was designed to require less frequent dosing and less peptide than T-20 and was assessed for its antifusogenic activity both in vitro and in vivo in the SCID-hu Thy/Liv mouse model. PC-1505 was essentially equipotent to the original C34 peptide and to T-20 in vitro. In HIV-1-infected SCID-hu Thy/Liv mice, T-20 lost activity with infrequent dosing, whereas the antiviral potency of PC-1505 was sustained, and PC-1505 was active against T-20-resistant ("DIV") virus with a G36D substitution in gp41. The in vivo results are the direct result of a significantly improved pharmacokinetic profile for the C34 peptide following albumin conjugation. Contrary to previous reports that the gp41 NHR trimer is poorly accessible to C34 fused to protein cargoes of increasing size (Hamburger, A. E., Kim, S., Welch, B. D., and Kay, M. S. (2005) J. Biol. Chem. 280, 12567-12572), these results are the first demonstration of the capacity for a large, endogenous serum protein to gain unobstructed access to the transient gp41 intermediates that exist during the HIV fusion process, and it supports further development of albumin conjugation as a promising approach to inhibit HIV-1 entry.

A covalent inhibitor targeting an intermediate conformation of the fusogenic subunit of the HIV-1 envelope complex.

Peptide inhibitors corresponding to sequences in the six helix bundle structure of the fusogenic portion (gp41) of the HIV envelope glycoprotein have been successfully implemented in preventing HIV entry. These peptides bind to regions in HIV gp41 transiently exposed during the fusion reaction. In an effort to improve upon these entry inhibitors, we have successfully designed and tested peptide analogs composed of chemical spacers and reactive moieties positioned strategically to facilitate covalent attachment. Using a temperature-arrested state prime wash in vitro assay we show evidence for the trapping of a pre-six helix bundle fusion intermediate by a covalent reaction with the specific anti-HIV-1 peptide. This is the first demonstration of the trapping of an intermediate conformation of a viral envelope glycoprotein during the fusion process that occurs in live cells. The permanent specific attachment of the covalent inhibitor is projected to improve the pharmacokinetics of administration in vivo and thereby improve the long-term sustainability of peptide entry inhibitor therapy and help to expand its applicability beyond salvage therapy.

Autocrine activation of the hepatocyte growth factor receptor/met tyrosine kinase induces tumor cell motility by regulating pseudopodial protrusion.

The multiple beta-actin rich pseudopodial protrusions of the invasive variant of Moloney sarcoma virus (MSV)-transformed epithelial MDCK cells (MSV-MDCK-INV) are strongly labeled for phosphotyrosine. Increased tyrosine phosphorylation among a number of proteins was detected in MSV-MDCK-INV cells relative to untransformed and MSV-transformed MDCK cells, especially for the hepatocyte growth factor receptor (HGF-R), otherwise known as c-met proto-oncogene. Cell surface expression of HGF-R was similar in the three cell lines, indicating that HGF-R is constitutively phosphorylated in MSV-MDCK-INV cells. Both the tyrosine kinase inhibitor herbimycin A and the HGFalpha antibody abolished HGF-R phosphorylation, induced retraction of pseudopodial protrusions, and promoted the establishment of cell-cell contacts as well as the apparition of numerous stabilizing stress fibers in MSV-MDCK-INV cells. Furthermore, anti-HGFalpha antibody abolished cell motility among MSV-MDCK-INV cells. Conditioned medium from MSV-MDCK-INV cells induced MDCK cell scattering, indicating that HGF is secreted by MSV-MDCK-INV cells. HGF titration followed by a subsequent washout of the antibodies led to renewed pseudopodial protrusion and cellular movement. We therefore show that activation of the tyrosine kinase activity of HGF-R/Met via an autocrine HGF loop is directly responsible for pseudopodial protrusion, thereby explaining the motile and invasive potential of this model epithelium-derived tumor cell line.