Drugs are designed to modulate the function of a critical protein involved in a disease; however, almost all small molecules have off-targets which can mitigate or altogether terminate their therapeutic efficacy. Unfortunately, there are no general methods that can identify all the protein targets that a drug binds to in an unbiased manner. For example, Histone Deacetylase Inhibitors (HDACIs) show promising clinical activity in many diseases; however, they are generally of low to moderate specificity and may act in part through, or be hindered by, uncharacterized off-target interactions. We propose a strategy that will allow for rapid and deep pharmacological profiling of early drug candidates that 1) identifies protein targets with extraordinary confidence, 2) localizes the precise site of interaction often with amino acid residue specificity, 3) is robust against metabolic alterations, 4) distinguishes the pharmacology of metabolites, 5) quantitates differences in pharmacological activity between cellular contexts. With this approach we will quantitate all interactions that each inhibitor has with proteins in a living cell. An neutron-encoded ?bar code? is added to activity-based probes during a click capture and release that allows us to blindly trace the drug in a nominal mass independent manner and simultaneously introduce quantitation channels. The barcodes are revealed in the isotopic fine structure by high resolution mass spectrometry yet do not compromise sensitivity at lower resolution fragmentation spectra. The neutron bar code is implemented by moving neutrons between elements and results in a prescribed pattern of relativistic nuclear mass defects embedded in the framework of a small molecule. With this method we can confidently retrieve drug-protein reaction products from in vivo systems regardless of metabolic alterations to the HDAC inhibitors, measure a pharmacological profile and determine molecular mechanism of action. Our central hypothesis is that neutron encoded activity based probe pharmacological profiling combined with innovative fragment-based discovery to generate selective HDACIs will enable the generation of a library of highly characterized and diversely selective HDACI probes. This will be realized through three specific aims:
in aim 1 we will employ a novel and systematically diverse group of sp3-enriched fragments to generate HDACIs that sample the rim region of HDACs leading to highly selective interactions.
In aim 2 we will measure the pharmacological profile of our novel HDACi?s and their effect on histone acetylation.
In aim 3, we will develop a multiplexed barcoding system which to enable higher detection and resolution of drug targets over the entire proteome. This overcomes many challenges universal to early stage drug development efforts that have frustrated the development of specific HDAC inhibitors in particular. Although HDACIs will be the general focus of this proposal our method is general for drug development in any area.
Drugs are designed to modulate the function of specific protein disease targets; however, there are currently no general methods for determining all off the unintended targets a drug may modulate leading to toxicity. In this project, we will develop a method designed to determine all the proteins to which a drug binds, which will enable chemists to develop compounds that engage the disease target with higher specificity. We will use this methodology to generate a new class of drug-like compounds that target an important class of cancer proteins; namely the histone deacetylases.
