netLibrary - eBook Summary Structure-based Drug Design by ...

Document
blood lymphocytes, peptide inhibition of IFN-τ antiretroviral activity implicated both the amino- and
carboxy-termini as functionally important [17].
Page 442
The structural basis of the antiproliferative activity of IFN-τ was also investigated. While multiple
regions were again involved in IFN-τ antiproliferative activity, it was the area adjacent to the carboxy
terminus, rather than the carboxy-terminus itself, which was the most crucial for antiproliferative
activity, inhibiting cell division by blocking entry into the S phase of the cell cycle [18]. Since, for a
particular IFN-α subtype, antiviral potency does not necessarily correlate with antiproliferative potency,
localization of these functions in different domains of the molecule is not unexpected [19]. Within all
known IFN-αs, the 8 amino acids from 139 to 147 are highly conserved. These residues are contained in
both carboxy-terminal peptides, but while they may be involved in antiviral activity, they do not appear
to be solely responsible for antiproliferative activity since the two peptides are not equivalent inhibitors
of IFN-τ antiproliferative activity. This observation is consistent with inhibition of antiviral activity but
not antiproliferative activity by a monoclonal antibody in this conserved region in human IFN-α and
with the requirement for tyrosine at position 123 for human IFN-α 1 antiproliferative activity [20,21]. It
has also been reported that mutations around Arg33 affected both antiviral and antiproliferative activity
of human IFN-α 4 on human cells [22], while the amino-terminus did not appear to be as important in
IFN-τ antiproliferative activity on bovine cells.
B. IFN-τ Monoclonal Antibodies
Another approach to structure/function analysis of IFN-τ involved generation of anti-IFN-τ monoclonal
antibodies. Four monoclonal antibodies were produced that reacted with the native IFN-τ protein. They
were epitope mapped using the available IFN-τ peptides. Two of the antibodies were directed against the
carboxy-terminus of the molecule, one against a region adjacent to the aminoterminus, and the final one
appeared to react with a conformational, rather than a linear determinant (C. Pontzer, unpublished data).
When these antibodies were used as competitors in binding assays, all four inhibited IFN-τ binding to
the Type I IFN receptor on MDBK cells. That anti-IFN-τ carboxy-terminal antibodies would inhibit
binding is not unexpected, but the inhibitory activity of the monoclonal antibodies directed against the
more amino-terminal region was not anticipated. There is the caveat that warns that results using
monoclonal antibodies to delineate function sites must be interpreted with caution since their size may
cause significant steric hindrance.
To proceed further with structural studies of IFN-τ, access to larger quantities of pure protein was
required. The obvious route to this end entailed production of recombinant protein. A synthetic gene for
IFN-τ was designed
http://legacy.netlibrary.com/nlreader/nlReader.dll?bookid=12640&filename=Page_442.html [4/9/2004 12:11:12 AM]