Residual Complexity Does Impact Organic Chemistry and Drug Discovery: The Case of Rufomyazine and Rufomycin

Abstract

Residual complexity (RC) involves the impact of subtle but critical structural and biological features on drug lead validation, including unexplained effects related to unidentified impurities. RC commonly plagues drug discovery efforts due to the inherent imperfections of chromatographic separation methods. The new diketopiperazine, rufomyazine (6), and the previously known antibiotic, rufomycin (7), represent a prototypical case of RC that (almost) resulted in the misassignment of biological activity. The case exemplifies that impurities well below the natural abundance of 13C (1.1%) can be highly relevant and calls for advanced analytical characterization of drug leads with extended molar dynamic ranges of >1:1,000 using qNMR and LC-MS. Isolated from an actinomycete strain, 6 was originally found to be active against Mycobacterium tuberculosis with a minimum inhibitory concentration (MIC) of 2 μg/mL and high selectivity. As a part of lead validation, the dipeptide was synthesized and surprisingly found to be inactive. The initially observed activity was eventually attributed to a very minor contamination (0.24% [m/m]) with a highly active cyclic peptide (MIC ∼ 0.02 μM), subsequently identified as an analogue of 7. This study illustrates the serious implications RC can exert on organic chemistry and drug discovery, and what efforts are vital to improve lead validation and efficiency, especially in NP-related drug discovery programs.

Isolation and Pharmacological Characterization of Six Opioidergic Picralima nitida Alkaloids

“…While the observed limited antinociceptive efficacy does not support akuamma's traditional use for pain relief, and is not congruous with a previous report of potent antinociception by 3 in rats, 19 this apparent contradiction is by far not unusual for investigations of ethnomedically used plants; as we and others have demonstrated, compound abundance and pharmacological relevance are not necessarily correlated, [35][36][37] and even very minor components or even impurities can be responsible for the observed biological activity. 38 Thus, while these studies provide detailed insight into the pharmacology of six highly abundant alkaloids present in the P. nitida extracts, other components present in lower abundance may possess potent antinociceptive activity. Furthermore, it is possible that pharmacokinetic and/or pharmacodynamic differences between rats and mice may account for the discrepant antinociceptive responses for the akuamma alkaloids in Wistar rats and C57BL/6 mice.…”

Natural products in drug discovery: advances and opportunities

“…In addition, microcrystal electron diffraction (MicroED) has recently emerged as a cryo-electron microscopy-based technique for unambiguous structure determination of small molecules 64 and is already finding important applications in NP research 65 . The increased resolution and sensitivity of analytical equipment can also help address problems associated with ‘residual complexity’ of isolated NPs; that is when biologically potent but unidentified impurities in an isolated NP sample (which could include structurally related metabolites or conformers) lead to an incorrect assignment of structure and/or activity 66 , 67 . To avoid futile downstream development efforts, Pauli and colleagues recommended that lead NPs should undergo advanced purity analysis at an early stage using quantitative NMR and LC–MS 67 .…”

Alkaloids in Contemporary Drug Discovery to Meet Global Disease Needs

“…Residual complexity becomes apparent when a very minor, undetected, component in a "pure" compound is responsible for the observed, potent activity. Two alkaloids serve as lead examples, sesbanine and sesbanimide [144,145], and rufomyazine and rufomycin [146]. There is also the attempt to trace the anti-TB activity of the highly purified, ubiquitous triterpene ursolic acid [147].…”