Fragment screening is becoming widely accepted as a technique to identify hit compounds for the development of novel lead compounds. In neighboring laboratories, we have recently, and independently, performed a fragment screening campaign on the HIV-1 integrase core domain (IN) using similar commercially purchased fragment libraries. The two campaigns used different screening methods for the preliminary identification of fragment hits; one used saturation transfer difference nuclear magnetic resonance spectroscopy (STD-NMR), and the other used surface plasmon resonance (SPR) spectroscopy. Both initial screens were followed by X-ray crystallography. Using the STD-NMR/X-ray approach, 15 IN/fragment complexes were identified, whereas the SPR/X-ray approach found 6 complexes. In this article, we compare the approaches that were taken by each group and the results obtained, and we look at what factors could potentially influence the final results. We find that despite using different approaches with little overlap of initial hits, both approaches identified binding sites on IN that provided a basis for fragment-based lead discovery and further lead development. Comparison of hits identified in the two studies highlights a key role for both the conditions under which fragment binding is measured and the criteria selected to classify hits.

CXCR4 is a G protein-coupled receptor with excellent potential as a therapeutic target for a range of clinical conditions including stem cell mobilization, cancer prognosis and treatment, fibrosis therapy and HIV. We report here the development of a fully human single-domain antibody-like scaffold termed an i-body, the engineering of which produces an i-body library possessing a long complementarity determining region (CDR) binding loop, and the isolation and characterisation of a panel of i-bodies with activity against human CXCR4. The CXCR4-specific i-bodies show antagonistic activity in a range of in vitro and in vivo assays including inhibition of HIV infection, cell migration and leukocyte recruitment but, importantly, not mobilization of hematopoietic stem cells. Epitope mapping of three CXCR4 i-bodies AM3-114, AM4-272 and AM3-523 revealed binding deep in the binding pocket of the receptor.

The single variable new antigen receptor domain antibody fragments (V_NARs) derived from shark immunoglobulin new antigen receptor antibodies (IgNARs) represent some of the smallest known immunoglobulin-based protein scaffolds. As single domains, they demonstrate favorable size and cryptic epitope recognition properties, making them attractive in diagnosis and therapy of numerous disease states. Here, we examine the stability of V_NAR domains with a focus on a family of V_NARs specific for apical membrane antigen 1 (AMA-1) from Plasmodium falciparum. The V_NARs are compared to traditional monoclonal antibodies (mAbs) in liquid, lyophilized and immobilized nitrocellulose formats. When maintained in various formats at 45 °C, V_NARs have improved stability compared to mAbs for periods of up to four weeks. Using circular dichroism spectroscopy we demonstrate that V_NAR domains are able to refold following heating to 80 °C. We also demonstrate that V_NAR domains are stable during incubation under potential in vivo conditions such as stomach acid, but not to the protease rich environment of murine stomach scrapings. Taken together, our results demonstrate the suitability of shark V_NAR domains for various diagnostic platforms and related applications.