Discovering anti-platelet drug combinations with an integrated model of activator-inhibitor relationships, activator-activator synergies and inhibitor-inhibitor synergies.

Federica Lombardi,Denis C Shields,Darren J Fitzpatrick,Niamh Moran,Fergal P Casey,Kalyan Golla,Donna K Slonim

doi:10.1371/journal.pcbi.1004119

Federica Lombardi, Denis C Shields + Show 5 more

Open Access

https://doi.org/10.1371/journal.pcbi.1004119

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Abstract

Identifying effective therapeutic drug combinations that modulate complex signaling pathways in platelets is central to the advancement of effective anti-thrombotic therapies. However, there is no systems model of the platelet that predicts responses to different inhibitor combinations. We developed an approach which goes beyond current inhibitor-inhibitor combination screening to efficiently consider other signaling aspects that may give insights into the behaviour of the platelet as a system. We investigated combinations of platelet inhibitors and activators. We evaluated three distinct strands of information, namely: activator-inhibitor combination screens (testing a panel of inhibitors against a panel of activators); inhibitor-inhibitor synergy screens; and activator-activator synergy screens. We demonstrated how these analyses may be efficiently performed, both experimentally and computationally, to identify particular combinations of most interest. Robust tests of activator-activator synergy and of inhibitor-inhibitor synergy required combinations to show significant excesses over the double doses of each component. Modeling identified multiple effects of an inhibitor of the P2Y12 ADP receptor, and complementarity between inhibitor-inhibitor synergy effects and activator-inhibitor combination effects. This approach accelerates the mapping of combination effects of compounds to develop combinations that may be therapeutically beneficial. We integrated the three information sources into a unified model that predicted the benefits of a triple drug combination targeting ADP, thromboxane and thrombin signaling.

Highlights

Cells are subject to diverse stimuli in vivo, and combine these inputs to generate appropriate biological responses
Drugs are often used in combinations, but establishing the best combinations is a considerable challenge for basic and clinical research
Platelets were incubated with inhibitors for 10 minutes at 37°C on orbital slow shake using a Wallac 1420 Multilabel Counter (Perkin Elmer). 10 μl cocktail (K) or activators were added and allowed to activate platelets for 10 minutes in the same conditions used with the inhibitors. 10 μl of the detection reagent Chrono-lume (Chronolog; Labmedics Limited, UK) were added and sample luminescence detected after an additional 5 seconds with rapid shaking measuring arbitrary absorbance units (AAU)

Summary

Introduction

Cells are subject to diverse stimuli in vivo, and combine these inputs to generate appropriate biological responses. Activators and inhibitors of various targets work together in different configurations to elicit valuable and sometimes unpredictable outcomes, both natural and therapeutically induced. Many therapeutic approaches combine multiple agents acting on different targets, for example in cardiovascular disease[1], cancer[2,3,4], and infection[5]. We would have a full systems model of every clinically important signaling process, helping us to predict and define potent combinations. Many workers seek to study the combination effects without considering additional information regarding the signaling network. Screens for novel agents can take a systematic approach[6,7], but are limited usually to comparing the inhibitor combinations to the effects of single agents, without considering wider aspects of the signaling system

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