NOT-OD-09-058 NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications Bacterial resistance to current classes of antibiotics is rapidly reaching a crisis point. Indeed, this has already become a primary cause of death in intensive care units of hospitals. Current classes of antibiotics, as a result of actual killing of the bacteria, create an evolutionary pressure for bacteria to evolve to escape their effects. An alternative approach has been proposed which targets bacterial virulence. In this approach, the goal is to inhibit specific bacterial functions that promote infection and are essential to persistence such as binding, invasion, subversion of host defenses, and chemical signaling. Such an approach has the advantage that inhibition of these functions will not kill the bacteria and therefore should result in a reduced selection pressure for drug-resistant mutations. This approach would also avoid the destruction of the normal host bacteria that is associated with current antibiotics. The enzymes DsbA and DsbB comprise the functional machinery in E. coli and many other bacteria to catalyze the formation of disulfide bonds in proteins in the periplasmic space. DsbA is the direct oxidant of protein substrates and resides in the periplasm. DsbB is an integral membrane enzyme which reoxidizes DsbA for further catalysis and transfers reducing equivalents to a bound quinone. We have recently solved the structure of DsbB using NMR spectroscopy and carried out extensive functional characterization. Intriguingly, disulfide bond formation is critical for the functioning of many proteins that mediate virulence functions, therefore the Dsb proteins have been suggested to be appropriate targets for the development of anti-virulence agents. Numerous studies have shown that mutants defective in the DsbA-DsbB oxidation system have reduced virulence in infection models. These results clearly support efforts to develop small molecule inhibitors of DsbB, as well as its functional homologs, as a novel approach to inhibit bacterial virulence. Based on this data, it is our hypothesis that small molecule inhibitors of DsbB will attenuate virulence functions of E. coli. To test this hypothesis, we are proposing in this competitive revision application the following new Specific Aim for addition to our grant titled """"""""Solution NMR Structure and Function of the Integral Membrane Protein DsbB"""""""":
Aim 1 : Optimization and testing of DsbB inhibitors. We will make a series of analogs of two hits we generated from a fragment screen against DsbB, one a quinone competitive inhibitor and one a mixed quinone/DsbA competitive inhibitor. These analogs will provide SAR and should make it possible to further optimize the potency of our lead compounds. Compounds will be tested using well-established UV or fluorescence assays for inhibitory activity. The most potent leads we generate will be tested for effects on DsbB function in vivo by looking at oxidation of a known substrate. Effects on virulence function will be assessed by examining effects on the motility of E. coli.
DsbB is an important protein for the functioning of many proteins that mediate the ability of bacteria to enter human cells, inject material into those cells, and to release toxins. All these functions contribute to the effects of bacterial infections on humans. Therefore, if we can inhibit the ability of bacteria to do this, we will have a new approach to treat bacterial infections. To that end, we are proposing to develop inhibitors of DsbB, a new potential approach to treating bacterial infections.
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| Früh, Virginie; Zhou, Yunpeng; Chen, Dan et al. (2010) Application of fragment-based drug discovery to membrane proteins: identification of ligands of the integral membrane enzyme DsbB. Chem Biol 17:881-91 |
| Zhou, Yunpeng; Cierpicki, Tomasz; Jimenez, Ricardo H Flores et al. (2008) NMR solution structure of the integral membrane enzyme DsbB: functional insights into DsbB-catalyzed disulfide bond formation. Mol Cell 31:896-908 |
