Figure 2: Inhibition of E. coli RelA by ppGpp, ppGpp-based compounds and Relacin derivatives.

The reaction mixture contained 30 nM RelA, 0.5 μM 70 S, 100 μM ppGpp, 0.3 mM [³H]GDP and 1 mM ATP. RelA enzymatic activity (turnover, ppGpp synthesized per RelA per minute) is normalized to that in the absence of an inhibitor. Error bars represent standard deviations of linear regression estimates, each experiment was performed at least three times. (a) ppGpp activates RelA at low concentrations (<50 μM) and acts as a weak inhibitor at higher concentrations. (b) Addition of N²-isobutyryl guanine base modification present in Relacin (highlighted in red) to DR-4250 yielding DR4239 does not dramatically alter the activity. Removal of 2′ hydroxyl group from DR-4250 yielding DR-6241A (Supplementary Table 1) and bis (phosphonoacyl) analogue DR-6331 yielding DR-5799C significantly decreases the activity. (c) Substitution of the base in ppGpp for adenine yielding ppApp or 6-thio-guanine yielding thio-ppGpp results in a dramatic increase in the efficiency of RelA inhibition (d) SAR elements characteristic for RelA inhibition by ppGpp-based inhibitors (b,c) are not transferable to Relacin scaffold: removal of the N²-isobutyryl protective group yielding di-iBu-Relacin compromises its activity against RelA and substitution of guanine for 6-thio-guanine yielding thio-di-iBu-Relacin leads to a complete inactivation.