Many cell-active compounds exert their phenotype by modulating the activity of several proteins at the same time – a phenomenon known as “polypharmacology”. Considering the number and complexity of expressed proteins in every cell it is not surprising that many bioactive compounds bind to multiple target proteins. Knowledge about these targets is important to understand the mechanism by which the drug exhibits its phenotype and can provide so-called biomarkers that can help to ensure that only patients that will benefit from treatment will receive this particular drug. Conversely, off-targets can lead to serious side-effects if not detected.
Our laboratory has established a suite of chemical biology and chemical proteomic approaches apt to reveal the molecular targets of small molecules by identifying proteins that bind to the compound in cells or cell extracts. Chemical proteomics combines affinity chromatography or protein thermal stability with mass spectrometry. In contrast to classical biochemical methods that use recombinant proteins, chemoproteomics is more unbiased as the full repertoire of natural proteins of a given cell is assessed at the same time and the experiment happens within a more physiological environment, i.e. a cell, rather than an isolated individual protein. We have also developed a technology called thermal stability profiling which evaluates changes in protein thermal stability due to target engagement in intact living cells using quantitative protein mass spectrometry.
The aim of the project is to profile small molecule drugs or drug candidates to understand their cellular targets enabling the potential identification of biomarkers, compound repurposing and/or the generation of more specific chemical probes.
The successful candidate will prepare functionalised affinity probes by means of medicinal chemistry and test them in cells and/or lysates of relevant cell lines or tissue using protein mass spectrometry. Bioinformatic analyses will be applied to identify the molecular targets thus enabling validation using classical molecular and cellular biology approaches. The work will be performed in close collaboration with the Proteomics group in the Target Discovery Institute. A specific focus will be on using patient material and exploring membrane targets.
Project reference number: 781
|Dr Kilian Huber||Structural Genomics Consortium||Oxford University, NDM Research Building||GBRemail@example.com|
|Professor Benedikt M Kessler||Target Discovery Institute||Oxford University, NDM Research Building||GBRfirstname.lastname@example.org|
Chemical proteomics provides a powerful means to gain systems-level insight into the mode of action of small molecules and/or natural products. In contrast to high-throughput screening efforts which only interrogate selected subproteomes such as kinases and often only consider individual domains, the methodology presented herein allows for the determination of the molecular targets of small molecules or drugs in a more relevant physiological setting. As such, the compound of interest is exposed to the entire variety of cellular proteins considering all naturally occurring posttranslational modifications and activation states. Samples prepared according to the procedures described in this protocol are compatible with lysates from cultured cell lines, primary cells, or samples from biopsies. In combination with state-of-the-art mass spectrometry techniques this approach grants access to a comprehensive view of small molecule-target protein interactions. Hide abstract
Thermal stabilization of proteins after ligand binding provides an efficient means to assess the binding of small molecules to proteins. We show here that in combination with quantitative mass spectrometry, the approach allows for the systematic survey of protein engagement by cellular metabolites and drugs. We profiled the targets of the drugs methotrexate and (S)-crizotinib and the metabolite 2'3'-cGAMP in intact cells and identified the 2'3'-cGAMP cognate transmembrane receptor STING, involved in immune signaling. Hide abstract
Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-β-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death. Hide abstract