Abstract
Purpose :
Previously, we validated using a destabilized E. coli dihydrofolate reductase (DHFR) domain to control the abundance of proteins of interest in the mouse eye. The DHFR-based destabilized domain (and virtually anything fused to it) undergoes rapid proteasome-mediated degradation in the absence of the antibiotic, trimethoprim (TMP). Presence of TMP (administered by drinking water, gavage or eye drops) stabilizes the DHFR fusion protein in a rapid and reversible manner. Yet, the extended use of a single antibiotic can disrupt the gut microbiota and can lead unnecessarily to antibiotic resistance. Herein we identified compounds that, like TMP, are able to control DHFR-YFP protein abundance in the eye, but do not act as antibiotics.
Methods :
Potential DHFR stabilizers were screened in HEK-293A and ARPE-19 cells expressing DHFR-YFP and/or YFP-DHFR using fluorescence microscopy and western blotting. Stabilizers were then evaluated for their ability to functionally control DHFR-based stress responsive signaling in stable cell lines. Next, a single lead compound was evaluated for antibiotic properties in E. coli cultures and in vivo in the mouse by analyzing the gut microbiota. The lead compound was then tested in vivo for its ability to stabilize DHFR-YFP (expressed by an rAAV2/2 MAX virus administered intravitreally) by fundus imaging and western blotting.
Results :
We identified a number of compounds that are able to stabilize N-terminal and C-terminal DHFR domains similarly to the canonical DHFR inhibitor, TMP. These compounds were non-toxic, bound to DHFR reversibly, and were able to control DHFR-based stress-responsive signaling identically to TMP. However, importantly, the identified lead compound from this series had no antibiotic activity at physiologically relevant levels, and had no effect on select commensal bacteria in the mouse gut (in contrast to TMP). Finally, the identified lead compound stabilized an ocular DHFR-YFP fusion with similar kinetics compared to TMP when administered by gavage.
Conclusions :
These non-antibiotic DHFR stabilizers should be considered as alternatives to TMP to minimize chances of developing antibiotic resistant bacteria and alterations in the gut microbiota while retaining the ability to stabilize potential DHFR-based gene therapy strategies.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.