Directed evolution of O6-alkylguanine-DNA alkyltransferase for applications in protein labeling

The specific reaction of O6-alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine (BG) derivatives allows for a specific labeling of AGT fusion proteins with chemically diverse compounds in living cells and in vitro. The efficiency of the labeling depends on a number of factors, most importantly on the reactivity, selectivity and stability of AGT. Here, we report the use of directed evolution and two different selection systems to further increase the activity of AGT towards BG derivatives by a factor of 17 and demonstrate the advantages of this mutant for the specific labeling of AGT fusion proteins displayed on the surface of mammalian cells. The results furthermore identify two regions of the protein outside the active site that influence the activity of the protein towards BG derivative

Directed evolution of O6-alkylguanine-DNA alkyltransferase for applications in protein labeling

The specific reaction of O6-alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine (BG) derivatives allows for a specific labeling of AGT fusion proteins with chemically diverse compounds in living cells and in vitro. The efficiency of the labeling depends on a number of factors, most importantly on the reactivity, selectivity and stability of AGT. Here, we report the use of directed evolution and two different selection systems to further increase the activity of AGT towards BG derivatives by a factor of 17 and demonstrate the advantages of this mutant for the specific labeling of AGT fusion proteins displayed on the surface of mammalian cells. The results furthermore identify two regions of the protein outside the active site that influence the activity of the protein towards BG derivatives

The anticancer natural product ophiobolin A induces cytotoxicity by covalent modification of phosphatidylethanolamine

Phenotypic screens allow the identification of small molecules with promising anticancer activity, but the difficulty in characterizing the mechanism of action of these compounds in human cells often undermines their value as drug leads. Here, we used a loss-of-function genetic screen in human haploid KBM7 cells to discover the mechanism of action of the anticancer natural product ophiobolin A (OPA). We found that genetic inactivation of de novo synthesis of phosphatidylethanolamine (PE) mitigates OPA cytotoxicity by reducing cellular PE levels. OPA reacts with the ethanolamine head group of PE in human cells to form pyrrole-containing covalent cytotoxic adducts and these adducts lead to lipid bilayer destabilization. Our characterization of this unusual cytotoxicity mechanism, made possible by unbiased genetic screening in human cells, suggests that the selective antitumor activity displayed by OPA may be due to altered membrane PE levels in cancer cells. DOI: http://dx.doi.org/10.7554/eLife.14601.00

A Novel Yeast-Based Platform for Small Molecule Target Deconvolution Reveals Previously Unknown Binding Partners for the Approved Drugs Erlotinib, Atorvastatin, and Sulfasalazine

The identification of all protein targets of a small molecule drug, i.e. target deconvolution, provides the basis for understanding its beneficial or deleterious actions. Target deconvolution remains a major and often problematic task within the biotechnology and pharmaceutical industries; there is therefore a generally acknowledged need for robust alternative methods to complement existing techniques. This thesis presents the development and application of a novel yeast-based platform for small molecule target deconvolution. The platform couples a sensitive yeast three-hybrid screening system for the identification of potential target proteins to an independent validation step based on affinity chromatography. The development of a novel yeast three-hybrid system was primarily based on the use of SNAP-tag to covalently anchor small molecules of interest onto a bait protein. The sensitivity of the system was improved by using an engineered reporter yeast strain to increase SNAP-tag labelling efficiency in living yeast cells. The reporter strain was engineered by knocking out genes encoding broad-specificity transporters from the yeast genome in order to reduce small molecule efflux from yeast cells. Such engineering was also necessary for the development of a negative selection strategy to reduce false positive levels in cDNA libraries. Finally, the system was judiciously optimised using defined small molecule-protein interactions of known affinity. In the second part of this work, the newly developed yeast three-hybrid system was applied to the screening of cDNA libraries for small molecule target deconvolution. Thirty-five small molecules, including well-characterised approved pharmaceutical drugs, were screened against eight different human cDNA libraries, resulting in the identification of forty-one small molecule-protein interactions. These screening results comprised both previously known and unknown interactions. The identification of numerous known targets of approved drugs validates the performance of the yeast-based approach for target deconvolution. Additionally, several of the previously unknown interactions were further validated using affinity chromatography and activity assays. Amongst these validated interactions, this work has uncovered the first non-kinase target of the epidermal growth factor receptor (EGFR) inhibitor erlotinib, i.e. oxysterol-binding protein-related protein 7 (ORP7); it has also led to the identification of off-targets of the popular HMG-CoA reductase inhibitor atorvastatin. The most significant finding of this thesis is that the widely used anti-inflammatory drug sulfasalazine is a potent inhibitor of sepiapterin reductase, an enzyme involved in the biosynthesis of the cofactor tetrahydrobiopterin. Despite its long use and clinical importance in the treatment of inflammatory bowel diseases and rheumatoid arthritis, its mechanism of action is not well understood. Here, it is proposed that sulfasalazine and/or its metabolite(s) inhibit sepiapterin reductase in vivo, this leading to a decrease in tetrahydrobiopterin biosynthesis and to an associated anti-inflammatory effect. This hypothesis provides an attractive explanation for some of the drug's properties and opens possibilities for new and improved therapies. In summary, this work establishes a powerful approach for small molecule target deconvolution and provides new insights into the mechanism of action of clinically used drugs

Compositions and use of sulfasalazine

The invention relates to compositions comprising sulfasalazine or sulfapyridine and products of a biosynthetic pathway that requires tetrahydrobiopterin. Such compositions are useful for the treatment of inflammatory bowel diseases or rheumatoid arthritis, but without the side effects observed with sulfasalazine alone. Furthermore the invention relates to compositions comprising sulfasalazine and 5 - aminosalicylic acid (mesalamine) for the treatment of the mentioned diseases. A further aspect of the invention is a method for monitoring the progress of sulfasalazine therapy comprising measuring the tetrahydrobiopterin level in body fluids of a patient under treatment with sulfasalazine. Yet another aspect of the invention is the use of sulfasalazine or sulfapyridine in a method of preventing pain in future treatment or in disease progression, such as delaying pain in cancer patients and preventing pain in surgery, dental care, chemotherapy or radiation therapy, preventing cardiogenic shock in acute myocardial infarction, preventing brain edema formation and intracranial hypertension in traumatic brain injury, and in a method of prevention or treatment of osteoporosis. All these various aspects of the invention are based on the observation that human sepiapterin reductase is a target of sulfasalazine and its metabolite sulfapyridine

Method of crosslinking two objects of interest

The invention provides a method of crosslinking two objects of interest, comprising the steps of : i) providing a fusion protein comprising at least a first protein and a second protein, wherein both the first and the second protein are, based on their structure and function, capable of forming a covalent bond with given substrates, and which first and second proteins are of substantially non-overlapping substrate selectivity, preferably of different substrate specificity ii) providing a first object of interest, comprising a substrate moiety for the first protein of the said fusion protein, and providing a second object of interest, comprising a substrate moiety for the second protein of the said fusion protein and iii) reacting said first protein of the fusion protein with the substrate moiety of said first object, and reacting said second protein of the fusion protein with the substrate moiety of said second object, thereby covalently crosslinking the first object to the second object via the said fusion protein. Most prominent applications of the disclosed method are, due to the straight-forward, reliable, directional and fast crosslinking reactions: the derivatization of cells, antibodies and the crosslinking of proteins