KRH-1636

We succeeded in restricting the conformation of a known CXCR4 ligand into a single active conformation by using a peptoid motif.In combination with structure-activity relationship studies the binding affinity was optimized to be 400 to 1500-fold higher than compounds currently under clin. development, such as KRH-1636 or AMD3100.AMD3100 is approved by the FDA for treatment of non-Hodgkin lymphoma and multiple myeloma.Together with the advantages of modified cyclic peptides as drugs (e.g. biocompatible metabolism, high stability against enzymic degradation, selectivity profile) this mol. has the potential to be a promising candidate for future medical applications in CXCR4 related diseases.

Affinity selection-mass spectrometry (AS-MS) is a sensitive technology for identifying small molecules that bind to target proteins, and assays enabled by AS-MS can be used to delineate relative binding affinities of ligands for proteins. 'Indirect' AS-MS assays employ size-exclusion techniques to separate target-ligand complexes from unbound ligands, and target-associated ligands are then specifically detected by liquid chromatography mass spectrometry. We report how indirect AS-MS binding assays with known reference control compounds were used as guideposts for development of an optimized purification method for CXCR4, a G-protein coupled chemokine receptor, for which we sought novel antagonists. The CXCR4 purification method that was developed was amenable to scale-up and enabled the screening of purified recombinant human CXCR4 against a large combinatorial library of small molecules by high throughput indirect AS-MS. The screen resulted in the discovery of new ligands that competed off binding of reference compounds to CXCR4 in AS-MS binding assays and that antagonized SDF1α-triggered responses and CXCR4-mediated HIV1 viral uptake in cell-based assays. This report provides a methodological paradigm whereby indirect AS-MS-based ligand binding assays may be used to guide optimal integral membrane protein purification methods that enable downstream affinity selection-based applications such as high throughput AS-MS screens.