Colloidal Aggregation and the in Vitro Activity of Traditional Chinese Medicines

Abstract

Traditional Chinese Medicines (TCMs) have been the sole source of therapeutics in China for two millennia. In recent drug discovery efforts, purified components of TCM formulations have shown activity in many in vitro assays, raising concerns of promiscuity. Here, we investigated 14 bioactive small molecules isolated from TCMs for colloidal aggregation. At concentrations commonly used in cell-based or biochemical assay conditions, eight of these compounds formed particles detectable by dynamic light scattering and showed detergent-reversible inhibition against β-lactamase and malate dehydrogenase, two counter-screening enzymes. When tested against their literature-reported molecular targets, three of these eight compounds showed similar reversal of their inhibitory activity in the presence of detergent. For three of the most potent aggregators, contributions to promiscuity via oxidative cycling were investigated; addition of 1 mM DTT had no effect on their activity, which is inconsistent with an oxidative mechanism. TCMs are often active at micromolar concentrations; this study suggests that care must be taken to control for artifactual activity when seeking their primary targets. Implications for the formulation of these molecules are considered.

Antibacterial Flavonoids from Medicinal Plants Covalently Inactivate Type III Protein Secretion Substrates

“…Our observations may explain some of the previously reported anti-infective properties of TCM extracts that produce active flavonoids 4,24 . As activated phenolic chemotypes present in baicalein and quercetin are also found in previous reported T3SS inhibitors (INP-0007 11 and TTS29 12 ) and other active plant metabolites from TCMs associated with protein aggregation 29 , the covalent modification of proteins described here may provide a general mechanism for inactivating proteins. Since many plant bacterial pathogens use type III secretion systems to cause disease and sense flavonoids 30 , it is possible that some of these compounds may have evolved as a consequence host-pathogen interactions.…”

The Essential Medicinal Chemistry of Curcumin

“…Further IMPS considerations include the PAINS characteristics of 1 , chiefly chemical aggregation, 46 the presence of a reactive Michael acceptor, 50 and fluorescence activity. This last attribute is particularly important as the main categories of reported activities in NAPRALERT (see also, Supporting Information Tables 1 and 2) emerge from cellular assays, which typically involve fluorescence and quenching for end point detection.…”

“…In addition to being unstable, it forms chemical aggregates (colloids) under common biochemical assay conditions. 46 Adding the nonionic detergent Triton X-100 attenuates the activity of 1 versus AmpC β-lactamase (IC 50 from 12 μM to >30 μM), malate dehydrogenase (IC 50 from 9 μM to >30 μM), and HIV-2 protease (IC 50 from 9 μM to >100 μM). This pattern is consistent with chemical aggregation and has been further confirmed by DLS (dynamic light scattering).…”

“…This pattern is consistent with chemical aggregation and has been further confirmed by DLS (dynamic light scattering). 46,78 These studies describe a critical aggregation concentration between 10 and 20 μM for compound 1 . This is highly relevant because it corresponds to common compound testing concentrations in many of the investigational assays we encountered.…”

Flavonoids: True or promiscuous inhibitors of enzyme? The case of deoxyxylulose phosphate reductoisomerase

“…Thus, according to the criteria established by Shoichet and co-workers, the tested flavonoids inhibit probably the enzyme via a non-specific mechanism. The method itself seems not questionable as it was already successfully used to prove the specificity of inhibition of some enzymes [34][35][36][37][38][39]. The phenomenon was especially noticeable for baicalein since no inhibition was observed in the presence of Triton X100.…”