The alarmone nucleotide (p)ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance and virulence, making (p)ppGpp-mediated signaling a promising target for development of antibacterials. Although ppGpp itself is an activator of the ribosome-associated ppGpp synthetase RelA, several ppGpp mimics have been developed as RelA inhibitors. However promising, the currently available ppGpp mimics are relatively inefficient, with IC50 in the sub-mM range. In an attempt to identify a potent and specific inhibitor of RelA capable of abrogating (p)ppGpp production in live bacterial cells, we have tested a targeted nucleotide library using a biochemical test system comprised of purified Escherichia coli components. While none of the compounds fulfilled this aim, the screen has yielded several potentially useful molecular tools for biochemical and structural work.

Department of Molecular Biology Umeå University Building 6K 6L University Hospital Area SE 901 87 Umeå Sweden

Graduate School of Science and Engineering Saitama University 255 Shimo Okubo Sakura ku Saitama Saitama 338 8570 Japan

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences v v i Flemingovo nám 2 166 10 Prague 6 Czech Republic

Laboratory for Molecular Infection Medicine Sweden Umeå University Building 6K and 6L University Hospital Area SE 901 87 Umeå Sweden

University of Tartu Institute of Technology Nooruse 1 50411 Tartu Estonia

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Direct detection of stringent alarmones (pp)pGpp using malachite green