Systematic discovery of multicomponent therapeutics

Abstract

Multicomponent therapies, originating through deliberate mixing of drugs in a clinical setting, through happenstance, and through rational design, have a successful history in a number of areas of medicine, including cancer, infectious diseases, and CNS disorders. We have developed a high-throughput screening method for identifying effective combinations of therapeutic compounds. We report here that systematic screening of combinations of small molecules reveals unexpected interactions between compounds, presumably due to interactions between the pathways on which they act. Through systematic screening of approximately 120,000 different two-component combinations of reference-listed drugs, we identified potential multicomponent therapeutics, including (i) fungistatic and analgesic agents that together generate fungicidal activity in drug-resistant Candida albicans, yet do not significantly affect human cells, (ii) glucocorticoid and antiplatelet agents that together suppress the production of tumor necrosis factor-alpha in human primary peripheral blood mononu-clear cells, and (iii) antipsychotic and antiprotozoal agents that do not exhibit significant antitumor activity alone, yet together prevent the growth of tumors in mice. Systematic combination screening may ultimately be useful for exploring the connectivity of biological pathways and, when performed with reference-listed drugs, may result in the discovery of new combination drug regimens.

Fig. 1.

Multicomponent therapeutics that prevent proliferation of fluconazole-resistant C. albicans. All 435 possible two-component combinations of 30 compounds were tested in duplicate in 36-point dose matrices to identify synergistic combinations. C. albicans cells were treated with compounds in 384-well plates and the extent of inhibition of Alamar blue fluorescence was determined. (A) Overview of synergistic combinations in C. albicans Alamar blue proliferation assay. Yellow columns and rows indicate compounds that are commonly used as antifungal agents. Purple squares indicate combinations for which neither compound is used as an antifungal agent on its own and red indicates combinations for which one, but not both, of the compounds is used as an antifungal agent on its own. (B) A sample combination dose matrix, showing the combined effect of pentamidine and phenazopyridine at six concentrations (including a zero concentration point) each. The experimentally measured inhibition of Alamar blue signal is shown for each pair of combinations. The color of the squares also indicates the level of Alamar blue inhibition. (C) The calculated excess inhibition over the predicted Bliss additivism model. The predicted Bliss additive effect (see text) was subtracted from the experimentally observed inhibition at each pair of concentrations. (D) The calculated excess inhibition over the HSA.

Fig. 2.

The combination of fluconazole and phenazopyridine selectively inhibits proliferation of fluconazole-resistant C. albicans.(A) The percent inhibition of fluconazole-resistant C. albicans proliferation is shown for the indicated concentrations of fluconazole and phenazopyridine, determined by using an Alamar blue proliferation assay. The average of three measurements is shown. (B) The calculated excess inhibition over the Bliss additivism model. The predicted Bliss additive effect (see text) was subtracted from the experimentally observed inhibition at each pair of concentrations. (C) The calculated excess inhibition over the HSA. (D) Percent inhibition of fluconazole-resistant C. albicans proliferation at concentrations showing optimal synergy [250 μM fluconazole (flu) and/or 20 μM phenazolepyridine (PAP)]. (E) Fungicidal activity, determined by using a cfu assay. Fluconazole-resistant C. albicans were treated with 20 μM PAP and/or 250 μM flu, or 4 μM amphotericin B (amp B) as a fungicidal positive control, and in each case an equal number of yeast particles were plated in the absence of any compound. The number of colonies that grew after each treatment regime is indicated. (F) The combination of PAP and flu prevents dye efflux. Fluconazoleresistant C. albicans cells were treated with 20 μM PAP and/or 250 μM flu and the effect on efflux of rhodamine G was determined by using fluorescence microscopy. Phase-contrast images of the yeast cells are shown in each case to confirm that a large number of cells were present in each case, but that dye efflux was only effectively inhibited when both PAP and flu were present. For all numerical figures shown, the average of three measurements is represented. Error bars, 1 SD.

Fig. 3.

Corticosteroids combined with dipyridamole (DP) selectively inhibit cytokine production. (A) Percent inhibition of TNF-α production in stimulated human peripheral blood cells (average of two measurements) using dexamethasone and dipyridamole at the indicated concentrations. (B) Excess inhibition over HSA for dipyridamole and dexamethasone. (C and D) Dipyridamole and dexamethasone inhibit production of TNF-α (C), but not IFN-γ (D). As a comparison, two steroids, fludrocortisone and prednisolone (E), were tested in combination for their effect on TNF-α production. In each case, the dilution factor is calculated based on the highest concentration tested [3 μM DP, 0.25 μM dexamethasone, 1 μM fludrocortisone (FLU), and 1 μM prednisolone (PRED)] for each agent, selected based on the empirically determined dose curves.

Fig. 4.

Chlorpromazine, an antipsychotic agent, and pentamidine, an antiprotozoal agent, together selectively prevent tumor cell growth in vitro and in vivo. (A) Percent inhibition of A549 proliferation, measured by using a BrdUrd incorporation assay (average of two measurements). (B and C) Excess antiproliferative activity over the Bliss additivism model (B) and the HSA (C). The predicted Bliss additive (B) or HSA (C) level of inhibition was subtracted from the level observed in A and plotted to indicate concentration regimes in which synergy is observed. (D) Effect of chlorpromazine and pentamidine on the growth of A549 lung carcinoma cell tumors in SCID mice. Tumor size as a function of time is plotted for each cohort and is treated as indicated. (E) The antiproliferative effect of the combination of chlorpromazine (chlor) and pentamidine (pent) was measured in human lung carcinoma cells (A549; ref. 21), in colon carcinoma cells (HCT116), and in normal human lung fibroblasts by using Alamar blue (46) to illustrate that higher concentrations are required for inhibition of normal fibroblasts relative to carcinoma cells.