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FRINATEK-Fri prosj.st. mat.,naturv.,tek

Chemical determinants of binding to ATP dependent enzymes and chemical library design

Awarded: NOK 7.0 mill.

Many cancers and other diseases may be treated by inhibiting enzymes that have become overactive: Human cells are composed mostly of proteins. These chains of hundreds to thousands of amino acid "building blocks", when joined, fold into specific shapes that enable their functions. Some of these, protein kinases, are phosphotransferase enzymes that transfer phosphate groups from ATP to other proteins. In doing so, they may create chain reactions of phosphate transfers and other reactions that together signal the cell to divide, or to die, or to survive, or to grow blood vessels, and so on. Cancer typically arises when mutations cause these signals to promote cancer growth and spreading. The atomic resolution structures of these enzymes greatly aid the design of therapeutic inhibitors, but complexities of enzyme-inhibitor interactions prevent exact design. As a result, multiple potential inhibitors must be designed, with a focus toward specific target enzymes. Inhibitor design is thus a hit-and-miss "shotgun" method, and better hit rates require better theoretical techniques for focussing. This project aimed to improve hit rates with detailed example studies of enzyme-inhibitor interactions, and apply the lessons to design new synthetic chemicals. The protein kinase enzymes studied included targets of leukemias, lung, and breast cancers, along with other protein kinases relevant to cell division and programmed cell death in general. The first area of study was of flexibilities of protein kinases. Enzymes are typically rigid, because they need specific shapes to stabilize high energy reaction intermediate structures to speed chemical reactions. However, protein kinases must be strictly controlled to prevent spurious signaling. Thus, protein kinases are usually disabled by structural changes that are in turn are controlled by chemical modification and protein complex formation. This has two major effects on drug discovery activities. First, inhibitors might bind to only one specific form of the enzyme. This means that some known protein structures may not be the structures that need to be targeted in patients. Second, the inhibitors themselves may cause the enzyme to change shape, so that the known protein structures may cause false predictions of poor binding of in fact good inhibitors. A second area of study involves the calculation of protein-inhibitor interaction energies. Proteins are too large to use the fundamental laws of physics (especially quantum mechanics) to calculate their properties. Even small proteins contain thousands of atoms, and these are tightly surrounded by water and other molecules, both large and small. Thus, theoretical methods must use severe approximations to predict the energies of protein-inhibitor interactions. One approach to compensate for the lack of a complete theoretical treatment is to fit the theory to each type of molecule, which can then be used to predict properties of similar molecules. Another approach is to avoid the use of model theories altogether, and try instead to extract from statistical data (using "machine-learning") the factors that determine binding energies, and use the results to design new molecules. Results from this project were presented in a series of lectures, posters, theses, and articles in international journals. Studies of the flexibility of the "glycine-rich loop" that covers ATP in protein kinases showed how crystal structures are more rigid than the kinases in solution (Oliveira et al, J. Phys. Chem 115, 2011), and how inhibitors may also bind with more than one conformation (Pflug et al., Acta Cryst F 68, 2012). The choice of crystal structures and methodology is critical for the type of inhibitors discovered (Gani et al., Chem. Biol. Drug Des. 82, 2013). Mutations involving protein kinases not only can cause cancer, they may change which inhibitors bind and cause drug resistance (Illert et al., Plos ONE 9, 2014), but this property may also be exploited for drug design purposes (Pflug et al., Biochem. J. 440, 2011; Åberg et al., Biol. Chem. 393, 2012). Understanding the structures and energies of inhibitor binding to their disease causing enzymes (Alexeeva et al., Acta Cryst D, 2015), along with predicting binding to related enzymes (causing either enhanced therapy or toxicity: Gani et al., BBA 1854, 2015; Martin et al., BBA 1854, 2015; Rothweiler et al, Eur J Med Chem 2015) enables the design of extremely potent inhibitors, especially when taking special interactions into account, such as those with sulfur, chlorine, or special chemical groups (Lauber et al., Chemistry 2015). Concluding these analyses, a set of chemical building blocks was synthesized to provide efficient starting points for rapid design of new inhibitors of new protein kinase targets for followup research projects.

"Structural genomics" research efforts have generally aimed to catalog a broad range of three dimensional structures of proteins. In contrast to this, efforts to understand the chemical recognition mechanisms that govern ligand-protein interactions--somet imes referred to as "chemogenomics"--require multiple detailed empirical studies of structures and flexibilities of closely related complexes. The Emil Fisher metaphor of "lock and key" to describe the enzyme - ligand complex captures the fact that the en zyme recognizes the right substance out of innumerable alternative possibility. However, the "lock and key" metaphor does not do justice to the fact that both "lock" and "key" are flexible, and "see" each other via a complex variety of chemical interactio ns, not simply by steric fit. This project aims to study these chemical interactions in detail for a selected set of key enzymes that require ATP as a substrate and that also represent targets for cancer drug design. From this understanding, libraries of potential ATP site binding ligands will be designed for drug discovery screening. Protein kinases are key signalling enzymes that are usually disregulated in cancers, and heat shock proteins are chaperones, or protein folding assistants, that also contrib ute to the uncontrolled survival of cancer cells. They share in common an ATP binding site, and therapeutic inhibitors of both classes are now emerging. This project focusses on studies of various inhibitors protein kinases A, B, and heat shock proteins H SP90 and HSP70, and variants thereof. The structures and flexibilities of the interactions will be studied by protein crystallography and calorimetric techniques. This will generate knowledge that can be systematized into algorithms for the design of new inhibitors for these and also related enzymes. Small molecules that represent potential fragments of these new inhibitors will be synthesized to create a library of substances useful for drug discovery.

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FRINATEK-Fri prosj.st. mat.,naturv.,tek