Target-agnostic SAR mapping and immunological evaluation of (-)-FR252921 and analogs against primary human immune...

The macrocyclic immunosuppressive natural product (-)-FR252921 was isolated in 2003 from a liquid culture of Pseudomonas fluorescens. Despite promising preliminary immunological insights, in-depth studies that advance our understanding of structure-activity relationship (SAR) have been scarce. Herein we document a detailed SAR mapping of (-)-FR252921 by the syntheses of 14 analogs and their subsequent evaluation. The composed library of analogs was designed to address crucial questions regarding the roles of explicit molecular features of the natural product while following a target-agnostic efficacy approach. Whereas previous efforts have focused on murine T cell assays, our endeavors transit to a primary human cell-based immunological platform using peripheral blood mononuclear cells (PBMCs), enabling direct assessment of immunosuppressive activity in B, natural killer (NK), and T cells via cytokine suppression. We emphasize that a new fully synthetic (-)-FR252921 analog (fs-FR4) profoundly outperformed the natural product in suppressing the response of both stimulated B cells (21-fold) and NK cells (16-fold) within the context the biological assays used, validating the power of our platform and standing as a lead candidate for further preclinical development. Lastly, we highlight that our studies included significant improvement in the synthetic efficacy of previously reported building blocks and key synthetic steps towards (-)-FR252921 and analogs.