Discovery of Selective Small-Molecule CD80 Inhibitors

Abstract

Protein-protein interactions are widely found in biological systems controlling diverse cellular events. Because these interactions are implicated in many diseases such as autoimmunity and cancer, regulation of protein-protein interactions provides ideal targets for drug intervention. The CD80-CD28 costimulatory pathway plays a critical role in regulation of the immune response and thus constitutes an attractive target for therapeutic manipulation of autoimmune diseases. The objective of this study is to identify small compounds disrupting these pivotal protein-protein interactions. Compounds that specifically blocked binding of CD80 to CD28 were identified using a strategy involving a cell-based scintillation proximity assay as the initial step. Secondary screening (e.g., by analyzing the direct binding of these compounds to the target immobilized on a biosensor surface) revealed that these compounds are highly selective CD80 binders. Screening of structurally related derivatives led to the identification of the chemical features required for inhibition of the CD80-CD28 interaction. In addition, the optimization process led to a 10-fold increase in binding affinity of the CD80 inhibitors. Using this approach, the authors identify low-molecular-weight compounds that specifically and with high potency inhibit the interaction between CD80 and CD28. These compounds serve as promising starting points for further development of CD80 inhibitors as potential immunomodulatory drugs. (Journal of Biomolecular Screening 2007:464-472)

Targeting B7‐1 in immunotherapy

“…Realizing the need to identify inhibitors that are active in the natural biological environment, another study employed a cell‐based scintillation proximity assay (SPA) as the primary screening tool to identify compounds for targeting the B7‐1:CD28 pathway. In this cell‐based SPA assay, B7‐1 was presented in a multivalent fashion on the SPA beads (by capture using Ig domain) and CD28 was expressed on the CHO cell lines, while a distance‐related energy transfer was monitored for discerning blockers and non‐blockers of the target interaction. Screening of a ligand library of 4000 compounds in this cell‐based assay resulted in one compound, named here as ligand 1 (Figure ), which inhibited 61% of the B7‐1:CD28 interaction at a 30 μM concentration .…”

“…In this cell‐based SPA assay, B7‐1 was presented in a multivalent fashion on the SPA beads (by capture using Ig domain) and CD28 was expressed on the CHO cell lines, while a distance‐related energy transfer was monitored for discerning blockers and non‐blockers of the target interaction. Screening of a ligand library of 4000 compounds in this cell‐based assay resulted in one compound, named here as ligand 1 (Figure ), which inhibited 61% of the B7‐1:CD28 interaction at a 30 μM concentration . Based on this initial hit, 150 analogs were generated and re‐screened using the SPA assay, which resulted in at least four compounds (named as ligand 2‐ligand 5 in Figure ), which showed improved activity in the SPA assay.…”

HTRF: A Technology Tailored for Drug Discovery - A Review of Theoretical Aspects and Recent Applications

“…It has also been used to study chemokine receptors (reviewed by Wang and Norcross [58]) and the interaction of surface molecules, such as Trail receptor 4 and OX40 [59], CD80 and CD28 [60], and adhesive molecule Chemokine (C-X3-C motif) ligand 1 ( CX3CL1 ) aggregation [61]. Moreover, the protein – peptide interaction was demonstrated with HTRF technology in cell surface calreticulin (CRT) and Shared Epitope (SE) from HLA-DR chain [62].…”

Identification of binding peptides of the ADAM15 disintegrin domain using phage display

“…Phage clones with a high affi nity for the target protein are identifi ed by DNA sequencing, and their corresponding amino acid sequences are determined. Phage display has been successfully used for the discovery of novel signalling proteins and small molecule inhibitors (Uvebrant et al 2007) such as the ADAM15-binding sorting nexin family member SNX30 (Karkkainen et al 2006).…”