Guimaraes, A; (2013) Screening molecular interactions for drug discovery. Doctoral thesis (PhD), UCL (University College London).

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Abstract

In biological systems, many proteins have specific binding sites for small-molecules or other proteins critical for their activity and function. Discovery of small-molecules that inhibit such protein interactions is useful in understanding and controlling protein function in disease. Hypoxia inducible factor (HIF1) is a heterodimeric transcription factor and its C-terminal activation domain (CTAD) interacts with the CH1 domain of p300 forming a complex known to regulate many genes. Spectral variants of green fluorescent protein were fused to the CTAD and CH1 to monitor the interaction between these proteins. Fluorescence resonance energy transfer (FRET) between these two chromophores occurred when the complex formed. A homogeneous screening assay was then developed for small-molecules with potential to inhibit the formation of the CTAD-CH1 complex. As part of the assay validation, some new small-molecule inhibitors previously tested by an alternative heterogeneous assay were found to inhibit within the same 100-500 μM concentration range. The new homogenous assay has promising potential for high-throughput screening of large chemical libraries. Novobiocin, a member of the aminocoumarin family can act as an antibacterial or anticancer agent. The clinical use of this class of antibiotics has been limited due to their low water solubility, low activity against gram-negative bacteria, and toxicity against cancer. Glycosyltransferases have been established as important tools in new drug development and are used here to improve water solubility and cell uptake. Glycosylation can be achieved enzymatically or chemically. A mass spectrometry based high-throughput screening (HTS) method was developed and used to find novel glycosylated aminocoumarins generated using a panel of glycosyltransferases and native/non-native sugar donors. The novobiocin derivatives were also re-synthesized chemically. The MIC for novobiocin in a DNA gyrase assay was 1 μM, and the derivatives showed similar MICs. However against a panel of human cancer cell lines these derivatives showed more than twice the activity.

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