Christopher Ullman

Repression of the HIV-1 5′ LTR promoter and inhibition of HIV-1 replication by using engineered zinc-finger transcription factors

Zinc finger domains are small DNA-binding modules that can be engineered to bind desired target sequences. Functional transcription factors can be made from these DNA-binding modules, by fusion with an appropriate effector domain. In this study, eight three-zinc-finger proteins (ZFPs) that bound HIV-1 sequences in vitro were engineered into transcription repressors by linking them to the Krüppel-associated box (KRAB) repressor domain (KOX1). One protein, ZFP HIVB-KOX, which bound to a 9-bp region overlapping two Sp1 sites, was found to repress a Tat-activated 5′ LTR cellular HIV-reporter assay to almost basal levels. A related six-finger protein, HIVBA′-KOX, was made to target all three Sp1 sites in the 5′ LTR promoter and efficiently inhibited both basal and Tat-activated transcription in unstimulated and mitogen-stimulated T cells. In contrast, a combination of two unlinked three-finger ZFPs, HIVA′-KOX and HIVB-KOX, which bind over the same region of DNA, resulted in less effective repression. Finally, HIVBA′-KOX was tested for its capacity to block viral replication in a cellular infection assay using the HIV-1 HXB2 strain. This ZFP was found to inhibit HIV-1 replication by 75% compared with control constructs, thus demonstrating the potential of this approach for antiviral therapy.

In vitro methods for peptide display and their applications

The presentation of recombinant peptide libraries linked to their coding sequence can be referred to as peptide display. Phage display is the most widely practiced peptide display technology but more recent alternatives such as CIS display, ribosome display and mRNA display offer advantages over phage for speed, library size and the display of unnatural amino acids. These have provided researchers with tools to address some of the failings of peptides such as their low affinity, low stability and inability to cross biological membranes. In this review, we assess some of the recent advances in peptide display and its application.

An in vitro selection strategy for conferring protease resistance to ligand binding peptides

One drawback to the use of peptides as therapeutics has been their susceptibility to proteolysis. Here, we have used an in vitro display technology, CIS display, to enhance the proteolytic resistance of ligand-binding peptides by selection of protecting motifs from a large peptide library. The premise to this selection was that certain linear peptides within a library could form structures capable of preventing the access of proteases to defined cleavage sites without affecting ligand binding. A diverse 12-mer peptide library was inserted between a FLAG epitope motif and a thrombin cleavage site and this construct was fused to the bacterial initiator protein RepA for CIS display selection. After five rounds of selection, protection motifs were isolated that were capable of preventing proteolytic cleavage of the adjacent thrombin site. Some of the selected peptides were also resistant to more promiscuous proteases, such as chymotrypsin and trypsin, which were not used in the selection. The observed resistance to thrombin, trypsin and chymotrypsin translated into increased resistance to plasma proteases in vitro and to an increase in circulating half-lives in rats. This method can be applied to enhancing the in vivo stability of therapeutic peptides.

Selection of a high-affinity WW domain against the extracellular region of VEGF receptor isoform-2 from a combinatorial library using CIS display

WW domains are small β-sheet motifs that are involved in intracellular signalling through the recognition of proline-rich or phosphorylated linear peptide sequences. Here, we describe modification of this motif to provide a framework for engineering the side chains exposed on its concave surface. This non-natural scaffold incorporates an additional tryptophan, has a shorter loop 1 and supports modification of 25% of the natural protein to form a novel affinity reagent. We demonstrate the utility of this structure by selecting a high-affinity binder to the extracellular region of human vascular endothelial growth factor receptor isoform 2 (VEGFR-2) from a library of modifications, using a cell-free molecular display platform, CIS display. The isolate has low nanomolar affinity to VEGFR-2 and inhibits binding of human VEGF to its receptor with nanomolar activity. The structure is amenable to cyclisation to improve its proteolytic stability and has advantages over larger protein scaffolds in that it can be synthesised chemically to high yields offering potential for therapeutic and non-therapeutic applications.