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  • Several other assay parameters were also explored First the

    2021-10-15

    Several other assay parameters were also explored. First, the thermal dependence of the assay was investigated by measuring the specific activity at different temperatures (Fig. 7). That data show that enzymatic activity increases with increasing temperature. Interestingly, the observed rate increased up to 45°C (the highest temperature attainable in our plate reader), suggesting that FDPS from E. coli and PFTase from S. cerevisiae are stable above their physiologically relevant temperatures of 37 and 30°C, respectively; this also indicates that this assay should be applicable for monitoring FDPS activity from mesophillic sources. Next, we wanted to explore whether this assay could be used to monitor FDPS activity in crude lysate. Accordingly, E. coli lysates were prepared and assayed for activity using the coupled assay. Activity was monitored for E. coli lysate derived from Colistin Sulfate that overexpressed the FDPS gene as well as for cells expressing only endogenous levels of FDPS. That data, illustrated in Fig. 8, show that FDPS activity can easily be detected in E. coli lysate even when the enzyme is present in impure form with no genetic manipulation to increase its level. It is useful to compare the sensitivity of this new assay with that of the aforementioned pyrophosphate release assay often employed for measuring FDPS activity. Data presented in Fig. 2 show that quantities of FPP product (converted to farnesylated dansyl-GCVIA) ranging from 100 to 200pmol can be easily detected using the PFTase-coupled assay. In contrast, because the pyrophosphate release assay employs UV detection in lieu of fluorescence, its limit of detection is much higher [12]; commercially available kits that use this assay claim a limit of detection of 1 to 2nmol [27]. Thus, this comparison suggests that the PFTase-based coupled assay is at least an order of magnitude more sensitive than the pyrophosphate release method. With appropriate reaction conditions defined from the above experiments, we next sought to examine the utility of this assay for screening inhibitors. Accordingly, three inhibitors (see Fig. 9), previously characterized using a spectrophotometric assay for phosphate releasing enzymes, were obtained and analyzed using the coupled enzyme assay developed here. Using Zoledronate, an IC50 value of 0.7μM was obtained (Fig. 10). That value compares favorably with the previously reported value of 1.1μM [11]. Similarly, values of 0.7μM for Risedronate and 11μM for Alendronate were obtained that also compare well with previously reported values. That data are summarized in Table 1. Control experiments showed that none of these compounds inhibited PFTase at any of the concentrations employed. Additional control experiments were performed where FPP was added to FDPS reactions containing Zoledronate present at saturating concentrations. With the addition of FPP, an increase in fluorescence was observed (data not shown), confirming that Zoledronate was not inhibiting PFTase. In a general sense, this experiment provides an easy method for verifying that a given inhibitor is a bone fide inhibitor of FDPS and is not an off-target artifact resulting from inhibition of the PFTase coupling enzyme instead. Finally, we analyzed the ability of the coupled assay to monitor FDPS inhibition by Zoledronate in E. coli lysate produced from cells overexpressing FDPS (Fig. 8, diamonds). Those experiments demonstrate that inhibition experiments can be performed with impure FDPS. More importantly, they suggest that this new coupled assay could be used to screen inhibitors present in complex biological samples including extracts containing natural products derived from animals, plants, or microorganisms.
    Conclusion The coupled enzyme assay described here provides a simple method to measure the rate of FDPS-catalyzed FPP production and can be used to measure IC50 values for inhibitors of that reaction. Relative to current methods, it has the significant advantage of providing a continuous optical readout and is hence amenable to implementation in a high throughput format; this new fluorescence-based assay is more sensitive than the currently existing coupled assay (that is based on absorbance). Thus, it should be useful for screening large libraries which in turn could lead to the development of new classes of inhibitors for this medically important target.