Impedance-based de-replication of antifungal drug classes 
High-throughput target-based drug screening of synthetic molecules so far has not resulted in new antifungal drug classes. One of the proposed reasons for this failure is the lack of penetration of synthetic molecules in the fungal cell, the high false-positive rates and the lack of selectivity. 
Three major classes of antifungal drugs are currently in use; the polyenes, the echinocandins and the azoles. Two of them, the polyenes and the echinocandins, are natural products or based on natural products produced by micro-organisms. Small molecules produced by micro-organisms have several desirable characteristics. First, they are highly specific due to their high degree of chirality. Second, they are intrinsically cell permeable and finally, they occupy a more diverse chemical space. However, natural product-based antifungal drug discovery is associated with a number of technical challenges. Fermentation extracts are composed of complex mixtures containing hundreds or thousands of unknown compounds and concomitantly natural product drug discovery is plagued by a high background of known and/or nuisance compounds. 
Recent efforts have revealed that the vast majority of micro-organisms are still unable to grow under laboratory conditions, indicating a huge unexplored source of new natural compounds. Mining this so-called ‘microbial dark matter’ has recently proven successful in the search for novel antibiotics, but has not been used in the search for novel antifungals. Even though previously uncultured micro-organisms can be a rich source of novel antimicrobials, they still produce known antimicrobials. Therefore, a high-throughput de-replication platform to filter out known or undesirable compounds would decrease costs and time consumption in antifungal drug discovery. 
We have developed a novel tool that can be used to de-replicate antifungal drug classes in a high-throughput and label-free fashion. This tool is based on multisine electrochemical impedance spectroscopy (EIS). Here, Candida albicans biofilms were grown on modified microtiter plates with gold electrodes coated at the bottom of the plates. Next, the cells were subjected to a small alternating potential inside the wells of the 96 well plates during biofilm development or destruction for 24h. The measurement of the resulting micro-current through the developing biofilm results in a signature profile based on multiple frequency components. When we challenged growing C. albicans biofilms with different antifungals we were able to obtain specific signatures for each class. By comparing the signature profile of unknown compounds with a library of signature profiles of known compounds we were able to identify the antifungal mode-of-action. 
Impedance based de-replication can become a valuable tool in the next-generation of natural product-based drug discovery platforms

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