Simplify: A Mass Spectrometry Metabolomics Approach to Identify Additives and Synergists from Complex Mixtures

Abstract

In fields ranging from environmental toxicology to drug discovery, it is critical to identify how multiple chemical compounds interact to perturb biological systems. Isolation-based approaches fail to incorporate multiconstituent interactions, such as synergy. We have developed an approach called "Simplify", which identifies mixture constituents that interact to achieve biological effects. Simplify combines biological and mass spectrometric data sets and uses an "activity index" to predict mixture interactions. Using the plant Salvia miltiorrhiza as a case study, we employed Simplify to identify four individual constituents that contribute to antimicrobial activity, three additives and one synergist. Our study is the first to enable identification of unknown synergists prior to isolating them, demonstrating the ability of the Simplify workflow to predict key contributors to the biological effect of a complex mixture. While utilized for natural products discovery in this study, this approach is expected to prove useful across multiple disciplines that rely on mixture analysis.

Figure 1.. S. miltiorrhiza compounds identified during this study.

Compounds 1-5 correspond to cryptotanshinone, dihydrotanshinone I, tanshinone IIA, 1-oxocryptotanshinone, and sugiol, respectively.

Figure 2.. Workflow for the Simplify Approach.

A known active compound is quantified in each complex mixture. An activity index (equation 1) is calculated by comparing the predicted and actual activity for each fraction. Fractions with activity indices greater than 110 are prioritized for follow up testing. Activity indices are used in SR modeling to predict constituents act additively or synergistically, which are subsequently isolated to confirm activity.

Figure 3.

Comparison of predicted to actual activity (A), black bars denote the activity due to cryptotanshinone and gray bars represent the observed fraction activity at 10 μg mL⁻¹. Cryptotanshinone’s MIC matches previous reports. Fractions SM-1, SM-3, and SM-5 were prioritized for synergy testing. SM-1 showed synergy with an ΣFIC of 0.38 (B) as did SM-3 with an ΣFIC of 0.19 (C). SM-5 is additive with an ΣFIC of 0.75 (D). Equation 2 was used to calculate ΣFICs.

Figure 4

A. Predicted and actual activities of third stage fractions resulting from chromatographic separation of S. miltiorrhiza fractions SM-3-2, SM-3-3, and SM-3-4 (see fractionation scheme in Figure S1) where black bars represent the predicted antimicrobial activity of each fraction due to cryptotanshinone and gray bars represent the actual activity of the fraction at 100 μg mL⁻¹. Cryptotanshinone served as a positive control, consistent with previous reports. B. Activity indices (equation 1) of fractions SM-3-2-1 through SM-3-4-5, where bars represent the extent to which each fraction enhances or suppresses the activity of cryptotanshinone. C. Selected dose response curves of cryptotanshinone with (black) and without (gray) 100 μg mL⁻¹ of synergistic (left), indifferent (middle), and additive fractions, corresponding to symbols in panel B.

Figure 5.. SR models used to predict additives and synergists.

High SRs correspond to m/z-retention time pairs that most likely possess activity. Each compound may be represented by more than one variable (i.e. isotopes and adducts). Compounds confirmed by NMR or MS-MS fragmentation are marked green, cryptotanshinone ions (compound 1) have been marked red, and unidentified variables have been marked blue. Compound 1 is not correlated with activity because it was spiked equally to all fractions. A. Additive SR plot using data from fractions SM-3-4-1 through SM-3-4-5. Dihydrotanshinone 1, tanshinone IIA, and 1-oxocryptotanshinone (compounds 2-4, respectively) were identified among the top ten additives. B. Synergistic SR plot using data from fractions SM-3-2-1 through SM-3-2-9. Sugiol (compound 5) was identified as a putative synergist

Figure 6.. Dose-response curves (± standard error) of cryptotanshinone, sugiol, and their combination.

Combined with cryptotanshinone, sugiol (inactive in isolation) causes a four-fold drop in cryptotanshinone’s IC₅₀ (5.89 to 1.56 μg mL⁻¹) illustrating synergy.