Exploring the Allosteric Inhibition of a MAPK Phosphatase Linked to Duchenne Muscular Dystrophy [electronic resource].

9781088318355

Ann Arbor : ProQuest Dissertations & Theses, 2019.

1 online resource (188 p.)

Access is available to the Yale community.

The development of mitogen-activated protein kinase (MAPK) phosphatases (MKPs) inhibitors has long been stymied by poor selectivity and bioavailability, leading these proteins to be considered "undruggable". Despite this, their role as regulators of the MAPKs makes them desirable therapeutic targets. As such, substantial effort has been placed in the identification of allosteric modulators of phosphatase activity, which avoid the pitfalls of active site-directed inhibition. MKP5 has recently been suggested as a target for the treatment for Duchenne muscular dystrophy (DMD), an inevitably fatal muscle wasting disease with significant unmet therapeutic need. To validate that MKP5 inhibition recapitulates the effects of genetic ablation, a high-throughput screen was carried out to identify a small molecule inhibitor of MKP5 by the Bennett Lab and the Yale Center for Molecular Discovery. Characterization of the hit compound emerging from this screen serves as the focus of this thesis. I have shown that the compound binds and inhibits MKP5 with micromolar potency while demonstrating strong selectivity relative to other MKPs. Kinetic experiments suggest that this compound inhibits by a mostly competitive mode of action. To further investigate this compound's mechanism of inhibition, I solved crystal structure of the hit compound in complex with the catalytic domain of MKP5. To our surprise, I found that the hit compound binds in a previously undescribed allosteric pocket located 8 A away from the site of catalysis. We identified a residue in this pocket, Tyr435, that is essential for both compound binding and enzymatic activity. Further, Met431 and Thr432 are residues unique to MKP5 that form hydrophobic interactions with the inhibitor and are likely integral to the compound's selectivity. In collaboration with the Ellman Lab and Jubilant BioSys, the structure-activity relationship and pharmacophore necessary for the inhibition of MKP5 was established by biochemical and structural studies. I solved the crystal structures of ten additional compounds in complex with MKP5, including a second compound from the high-throughput screen that binds in the allosteric pocket despite containing a distinct chemical scaffold. This work culminated in the identification of a sub-micromolar inhibitor which improves on the initial hit in both potency and solubility. The Bennett Lab showed that inhibition of MKP5 in mouse myoblasts and fibroblasts leads to the increased activation of MAPK substrates of MKP5, promotes myogenesis, and limits profibrotic signaling by reducing the activation of the transforming growth factor β effector protein Smad2. In this work, we have revealed a new strategy for the inhibition of MKP5 and made great strides toward the validation of MKP5 as a valid pharmacological target for the treatment of DMD.

Dissertations & Theses @ Yale University.

Books / Online / Dissertations & Theses

English

January 17, 2020

Thesis (Ph.D.)--Yale University, 2019.

Yale University. Pharmacology.