A multifunctional NMR and fluorescence-based chemical probe for dehydrogenases: Application to an anti-infective drug target.

摘要

Dihydrodipicolinate Reductase (DHPR) is a potential anti-infective drug target. Chapter 1 described how to express, purify and characterize this protein target in an unlabeled and a triple labeled form, for later studies. A privileged scaffold catechol rhodanine acetic acid (CRAA) is a starting point to design dehydrogenase inhibitors. This privileged scaffold has also been used to develop proteomic probes based on its fluorescent/visible properties. It was developed to use in in-gel competition assays for dehydrogenases in Chapter 2. In Chapter 3, this CRAA scaffold was linked to an agarose matrix to build an affinity column to 'fish' out possible dehydrogenases from the Mycobacterium Tuberculosis proteome, to identify potential drug targets, and human liver proteomes, to identify potential anti-targets, which should be avoided in the design of anti-infective drug leads. The mechanism by which DHPR binds to its cofactor and substrate was studied in Chapter 4; this mechanism facilitated an understanding of ligand cooperativity for DHPR, and the design of high affinity biligand inhibitors. The 15N-carboxamide labeled NAD(H) was developed and applied as a mechanistic probe for dehydrogenases in Chapter 5, these probes can help us to better understand the catalytic mechanism and the cofactor carboxamide geometry, when they bind to dehydrogenases. In Chapter 6, fragment assembly drug design methods are applied using NMR and thiol tethering approaches, developed based on the DHPR-CRAA-PDC complex that was characterized in chapter 4. Labeled DHPR and cofactor were prepared to permit a better understanding of protein-ligand interaction mechanisms, and to validate the high throughput screening. Successful results from this study will facilitate future rational drug design using fragment assembly.