Vancomycin-resistant enterococci (VRE) have become a major cause of nosocomial infections and represent a serious public health threat. Limited therapeutic options are available to combat VRE infections, and therefore substantial efforts have been invested in modifying vancomycin to produce new forms of the drug that can overcome these antibiotic-resistant pathogens. However, these efforts have met with limited success, in large part because we do not understand the molecular mechanisms of action that allow certain vancomycin variants to be active against VRE, while others are not. In VRE, vancomycin resistance results from acquisition of a gene cluster encoding enzymes that remodel the bacterial cell wall. The remodeling alters the binding epitope for vancomycin, reducing the antibiotic's affinity for its target 1000-fold. Many investigators have modified vancomycin in bids to restore activity against VRE; commonly, efforts have centered around constructing dimeric or multivalent forms of the drug, with the expectation that they will be able to use avid binding to overcome low affinity and improve binding of the target. However, the results have been mixed, with some vancomycin dimers showing activity against VRE, while others do not, indicating that the avid binding model is inadequate to explain the functioning of these molecules. This lack of a mechanistic framework stymies rational development efforts and hampers one of the most promising avenues to new treatments for VRE infections. To overcome this hurdle, we propose in our first aim a rigorous set of functional, mechanistic, and structural studies aimed at uncovering the mechanism(s) of action that confer anti-VRE activity on certain vancomycin dimers. We have chosen a representative panel of vancomycin dimers that have demonstrated strong antimicrobial activity with VRE strains, and will elucidate the contribution of avid binding to their activity, the structural basis of their target recognition, and whether other mechanisms contribute to their effects. In the second aim we propose a complementary approach, building upon emerging results that have demonstrated that ligands prompt vancomycin to assemble into a higher-order supercomplex. Following up on our structural characterization of this supercomplex, we have designed structure-based crosslinking strategies to stabilize supercomplex assembly, and will use them to test the effects of supercomplex formation upon target recognition and activity against VRE strains. The mechanistic insights provided by these experiments will enable future rational development of new therapeutics to combat VRE.
Vancomycin-resistant enterococci (VRE) have become important causes of hospital-acquired infections and now represent serious threats to public health. Substantial efforts have been devoted to developing new forms of vancomycin that can be used to control VRE, but these efforts have enjoyed only limited success, in large part because we do not yet understand the molecular mechanisms by which a therapeutic can overcome VRE. We propose a rigorous set of functional, mechanistic, and structural studies to uncover precisely how vancomycin modification can be used to target VRE; this information will prove invaluable in the rational development of new antibiotics to combat this emerging crisis.
