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Title Developing Kinase Inhibitors as Chemical Tools and Potential Drugs.
Publication Date
Date Accessioned
Degree PhD
Discipline/Department Medicinal Chemistry
Degree Level doctoral
University/Publisher University of Michigan
Abstract Kinase inhibitors have experienced a dramatic evolution over the last two decades, as multi-targeted kinase chemical probes have gradually given way to highly specific, clinically approved drugs. Recent advances in molecular targeted therapies, especially those involved in cancer, have also created a demand for novel inhibitors that can potently and selectively target any of the more than 200 kinases implicated in disease. The work described herein details our efforts toward designing these needed methodologies. Our first strategy utilized covalent kinase inhibition to target a nonconserved cysteine residue located within the phosphate-binding loop (P-loop) of c-Src kinase. These covalent inhibitors displayed improved potency and selectivity for the target over their reversible counterparts. Continuation of this initial study allowed us to characterize the relative conformation of the kinase P-loop as a means for selective inhibition. The P-loop is a structural feature implicated in inhibitor selectivity and drug resistance, yet it is often not resolved in kinase X-ray crystal structures. The synthesis of a unique ‘molecular ruler’ series of covalent compounds resulted in an unprecedented tool to measure the native, in-solution positioning of this critical loop. We also designed a proximity-based, enzyme-templated fragment screen for the discovery of selective bivalent kinase inhibitors. This approach rapidly identified fragment hits without the need for biophysical detection methods or prior structural knowledge and could potentially be applied to non-kinase proteins. Our final targeting approach evaluated the impact that conformation-selective inhibition has on kinome-wide selectivity. A crystallographically confirmed ‘matched set’ of active and inactive inhibitors sharing the same kinase hinge-binding scaffold was used to determine trends in inhibitor selectivity. We observed that DFG-out inactive (type II) kinase inhibitors experienced an identical selectivity score to dasatinib, an inhibitor that binds to the active conformation of the kinase. Conversely, our designed C-helix out inactive (CHO) inhibitors produced a far superior selectivity profile. All of these methods have greatly improved our knowledge regarding kinase inhibitor selectivity, which should aid in the development of future chemical probes and clinical drug candidates.
Subjects/Keywords Kinase inhibitor; Src kinase; Biological Chemistry; Science
Contributors Soellner, Matthew Bryan (committee member); Garcia, George A (committee member); Mapp, Anna K (committee member); Brooks III, Charles L (committee member); Martin, Brent Randall (committee member)
Language en
Country of Publication us
Record ID handle:2027.42/120774
Repository umich
Date Retrieved
Date Indexed 2019-06-03
Grantor University of Michigan, Horace H. Rackham School of Graduate Studies
Issued Date 2016-01-01 00:00:00
Note [thesisdegreename] PhD; [thesisdegreediscipline] Medicinal Chemistry; [thesisdegreegrantor] University of Michigan, Horace H. Rackham School of Graduate Studies; [bitstreamurl] http://deepblue.lib.umich.edu/bitstream/2027.42/120774/1/fkw_1.pdf;

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…Alignment of crystal structures with dasatinib bound to c-Src (PDB: 3QLG) and c-Abl (2GQG). 26 2.5 Relative kinact values for compound 2.7. 27 3.1 Surface map representation of kinase catalytic domain. 51 3.2 Comparison of…

…traditional tethering (left) to our hybrid approach (right). 52 3.3 Structures of promiscuous kinase inhibitors 2.1, 3.1 and 3.2. 53 3.4 Graph of enzyme-templated screen using 3M c-Src and a library of 110 acrylamides (left)…

…54 3.5 Structures of bivalent kinase inhibitors. 55 xi 3.6 Illustration of three critical residues involved in the extended P-loop conformation of c-Src (PDB: 2SRC). 58 4.1 DFG-out dasatinib analog characterization. 83 4.2 αC…

…helix out dasatinib analog characterization. 84 4.3 Dasatinib analog BODIPY data. 88 4.4 Dasatinib analog kinome-wide selectivity. 89 5.1 Depiction of kinase P-loop. 116 5.2 P-loop overlay for c-Src (blue) and Hck (green)…

…wt, V313I, and V377I c-Src. 131 A.1 Kinase P-loop sequence alignment. 158 A.2 Compound 2.1 KINOMEscan profiling results. 158 A.3 Gel filtration results with irreversible inhibitors. 159 A.4 Mass spectrometry results of enzyme-inhibitor…

…Overlay of DAS-DFGO-I and imatinib. 242 C.3 DAS-CHO analog biochemical IC50 curves. 243 C.4 DAS-BODIPY and DAS-DFGO-II-BODIPY phospho-Src data. 244 D.1 PP2 binding to c-Src kinase. 291 D.2 Kinase P-loop sequence alignment. 291 D.3 Inhibitor…

…herein details our efforts toward designing these needed methodologies. Our first strategy utilized irreversible kinase inhibition to target a nonconserved cysteine residue located within the phosphate-binding loop (P-loop) of c-Src kinase. A…

…promiscuous kinase inhibitor scaffold served as the design basis, providing for a stringent test of inhibitor selectivity. Lead covalent compounds, 2.6 and 2.7, both displayed an 8-fold improvement in potency for c-Src over a reversible analog, compound 2.1…