Spirolactam rhodamines are fluorescent photoswitches that are useful for single molecule localization microscopy, volumetric 3D digital light photoactivatable dye displays, and other applications. Measurement of the photophysical properties, particularly photoswitching kinetics and quantum yields, is challenging and a comprehensive understanding of how molecular structure affects these parameters remains incomplete. In this study, we have synthesized a series of N‐aryl spirolactam rhodamine photoswitches with fluorescent emissions at 585 nm and 518 nm. Extinction coefficients and fluorescence quantum yields of the fluorescent form of the photoswitch have been measured using excess trifluoroacetic acid to drive the equilibrium to the open form. A method to determine photoswitching kinetics and quantum yields was developed by monitoring the kinetics to reach equilibrium between the on‐state and off‐state and fitting this data to a rate equation for a reaction in equilibrium. Trends based on the electronic and steric properties of the aryl substituents were evaluated. Using this information, a proof‐of‐principle demonstration of 3D voxel formation was accomplished using a green (518 nm) emitting photoswitch, setting the foundation for a multi‐colour volumetric 3D display.

Overexpression of ABC transporters like P-glycoprotein (P-gp) has been correlated with resistances in cancer chemotherapy. Intensive efforts to identify P-gp inhibitors for use in combination therapy have not led to clinically approved inhibitors to date. Here, we describe computational approaches combined with structure-based design to improve the characteristics of a P-gp inhibitor previously identified by us. This hit compound represents a novel class of P-gp inhibitors that specifically targets and inhibits P-gp ATP hydrolysis while not being transported by the pump. We describe here a new program for virtual chemical synthesis and computational assessment, ChemGen, to produce hit compound variants with improved binding characteristics. The chemical syntheses of several variants, efficacy in reversing multidrug resistance in cell culture, and biochemical assessment of the inhibition mechanism are described. The usefulness of the computational predictions of binding characteristics of the inhibitor variants is discussed and compared to more traditional structure-based approaches.

ATP‐binding cassette (ABC) transporters are transmembrane proteins that act as drug efflux pumps and are associated with multi‐drug resistance (MDR) in cancers. Two specific proteins that belong to this protein superfamily are P‐glycoprotein (P‐gp) and breast cancer resistant protein (BCRP). These two proteins are known to pump chemotherapeutics out of cells, rendering treatments of some cancers less efficient. Certain cancer cell lines overexpress these proteins, leading to MDR. If a compound can be identified that inhibits these efflux pumps, then sensitivity to chemotherapeutics can be restored. The levels of expression of P‐gp and BCRP were quantitatively measured in four cancer cell lines that are used by us to screen for inhibitors of P‐gp or BCRP. The expression of P‐gp, BCRP, and MRP1 (multidrug resistance‐associated protein 1) was measured using quantitative One‐Step qPCR in a prostate cancer cell line, DU145, and compared to an MDR derivative line that overexpresses P‐gp, DU145 TXR. We found that DU145 TXR expresses P‐gp, BCRP, and MRP1 by about 14,000‐fold, 4‐fold, and 0.75‐fold, respectively, when compared to the parental cell line, DU145. A breast cancer cell line, MCF‐7, and its mitoxantrone‐resistant derivative, MCF‐7 M100, were also analyzed. The MCF‐7 M100 cell line was found to express BCRP, P‐gp, and MRP1 by about 600‐fold, 0.3‐fold, and 1.6‐fold, respectively, compared to the parental cell line, MCF‐7. The results were qualitatively confirmed at the protein level using Western Blot analyses. In other experiments, we demonstrated that P‐gp and BCRP inhibitors identified by us that reversed multidrug resistances in the MDR prostate and breast cancer cell lines, DU145 TXR and MCF‐7 M100, did not down‐regulate expression levels of P‐gp or BCRP, suggesting that reversal of MDR was accomplished by inhibiting transport processes and not transporter expression levels.Support or Funding InformationThis work is supported by NIH NIGMS [R15GM09477102] to John G. Wise, SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas, as well as the Hamilton Undergraduate Research Scholars Program and the SMU Summer Research Assistantship and Undergraduate Research Assistantship Programs.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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Multidrug resistance (MDR) in cancer and other diseases is frequently associated with transmembrane efflux proteins, one of which is P‐glycoprotein (P‐gp). Over‐expression of P‐gp in cancer cells increases the efflux of therapeutic drugs rendering them ineffective. In order to re‐sensitize multidrug resistant cancers to chemotherapy, we have found inhibitors of P‐gp by in silico screening methods (Brewer et al., Molecular pharmacology 86, 716–726, 2014), and several of these inhibitors were shown to successfully overcome MDR in the drug resistant DU145TXR prostate cancer cell line (Follit et al., Pharmacol. Res. Perspect. 3, e00170, 2015). In this study, we expanded our cell culture screening to evaluate 25 new compounds identified by our in silico screening method and report that over 30% of these compounds re‐sensitized DU145TXR cancer cells to chemotherapy, while not exhibiting significant toxicity to the non‐cancerous human lung fibroblast (HFL‐1) cell line. The best of the identified hit compounds were further investigated with regard to the mechanism of inhibition using ATPase activity assays and electron spin resonance spectroscopy (ESR). Several of the best compounds were found to inhibit ATPase activity. Using a spin‐labeled ATP analog and ESR spectroscopy, we assessed the effects of the identified inhibitors on ATP binding to P‐gp. Variants of one of the original hit compounds, SMU 29, that were computationally predicted to interact more strongly with P‐gp have been synthesized and were analyzed here for their effects on P‐gp ATPase activity and ATP binding characteristics.Support or Funding InformationThis work is supported by NIH NIGMS [R15GM094771‐02] to John G. Wise, SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas, as well as the Hamilton Undergraduate Research Scholars Program and the SMU Summer Research Assistantship and Undergraduate Research Assistantship Programs.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.