There are remarkably few examples of designing cyclic constraints for peptides which adopt an irregular conformation in their bioactive state. We discuss the design strategies, opportunities and challenges for this underexplored class of inhibitors.

Mannich base PIP-199 is the only reported smallmolecule inhibitor of the Fanconi anemia complementation group M-RecQ-mediated genome instability protein (FANCM-RMI), a protein−protein interaction that governs genome instability in the genetic disorders Fanconi anemia and Bloom's syndrome. PIP-199 and analogues with the same indole-derived Mannich base scaffold have been used as tool compounds in diverse biological studies. We report the first published synthesis of PIP-199 and its analogues, demonstrating that PIP-199 immediately decomposes in common aqueous buffers and some organic solvents. Neither PIP-199 nor its more hydrolytically stable analogues show any observable activity in binding and competitive biophysical assays for FANCM-RMI. We conclude that PIP-199 is not an effective tool compound for biological studies and that apparent cellular activity likely arises from the nonspecific toxicity of breakdown products. More generally, apparent inhibitors that share this Mannich scaffold potentially represent a new family of pan-assay interference compounds (PAINS) that should be thoroughly assessed for aqueous stability prior to use in biological studies.

PIP-199 is the only reported small molecule inhibitor of FANCM-RMI, a key protein-protein interaction that governs genome instability in the genetic disorders Fanconi Anemia and Bloom’s Syndrome. PIP-199 and close analogues that share the same indole-derived Mannich base core scaffold have been used as commercially available tool compounds for studying a wide range of therapeutically relevant biological pathways and targets, including the Alternative Lengthening of Telomeres (ALT), G-quadruplexes, pro-apoptotic proteins, and quorum sensing. Herein, we report the first published synthesis of PIP-199 and related analogues, demonstrating that the parent compound immediately decomposes in common aqueous buffers and some organic solvents. We characterize the breakdown products and show that PIP-199 and its more hydrolytically stable analogues show no observable activity in binding and competitive biophysical assays for FANCM-RMI. We conclude that PIP-199 is not an effective tool compound for biological studies, and that apparent activity in cellular studies likely arises from non-specific toxicity of mixed breakdown products. More generally, apparent inhibitors that share this indole-derived Mannich scaffold potentially represent a new family of pan-assay interference compounds (PAINS) that should be thoroughly assessed for aqueous stability prior to use in biological studies.

PIP-199 is the only reported small molecule inhibitor of FANCM-RMI, a key protein-protein interaction that governs genome instability in the genetic disorders Fanconi Anemia and Bloom’s Syndrome. PIP-199 and close analogues that share the same indole-derived Mannich base core scaffold have been used as commercially available tool compounds for studying a wide range of therapeutically relevant biological pathways and targets, including the Alternative Lengthening of Telomeres (ALT), G-quadruplexes, pro-apoptotic proteins, and quorum sensing. Herein, we report the first published synthesis of PIP-199 and related analogues, demonstrating that the parent compound immediately decomposes in common aqueous buffers and some organic solvents. We characterize the breakdown products and show that PIP-199 and its more hydrolytically stable analogues show no observable activity in binding and competitive biophysical assays for FANCM-RMI. We conclude that PIP-199 is not an effective tool compound for biological studies, and that apparent activity in cellular studies likely arises from non-specific toxicity of mixed breakdown products. More generally, apparent inhibitors that share this indole-derived Mannich scaffold potentially represent a new family of pan-assay interference compounds (PAINS) that should be thoroughly assessed for aqueous stability prior to use in biological studies.