Bisphosphonate drugs (e.g., Fosamax and Zometa) are thought to act primarily by inhibiting farnesyl diphosphate synthase (FPPS), resulting in decreased prenylation of small GTPases. Here, we show that some bisphosphonates can also inhibit geranylgeranyl diphosphate synthase (GGPPS), as well as undecaprenyl diphosphate synthase (UPPS), a cis-prenyltransferase of interest as a target for antibacterial therapy. Our results on GGPPS (10 structures) show that there are three bisphosphonate-binding sites, consisting of FPP or isopentenyl diphosphate substrate-binding sites together with a GGPP product-or inhibitor-binding site. In UPPS, there are a total of four binding sites (in five structures). These results are of general interest because they provide the first structures of GGPPSand UPPS-inhibitor complexes, potentially important drug targets, in addition to revealing a remarkably broad spectrum of binding modes not seen in FPPS inhibition.cell wall ͉ geranylgeranyl diphosphate synthase ͉ undecaprenyl diphosphate synthase ͉ x-ray structure I soprenoid biosynthesis involves the condensation of C 5 -diphosphates to form a very broad range of compounds used in cell membrane (cholesterol, ergosterol), cell wall (lipid I, II, peptidoglycan) and terpene biosynthesis, electron transfer (quinone, heme a, carotenoid, chlorophyll), and in many eukaryotes, cell signaling pathways (Ras, Rho, Rap, Rac). There has, therefore, been considerable interest in developing specific inhibitors of some of these pathways to modify cell function. For example, the bisphosphonate drugs used to treat bone resorption diseases such as osteoporosis (1) have been thought to function by targeting farnesyl diphosphate synthase (FPPS, EC 2.5.1.10) in osteoclasts, leading to dysregulation of cell-signaling pathways involving small GTPases, and in some parasitic protozoa, leading to inhibition of ergosterol biosynthesis (2). However, in recent work Goffinet et al. (3) proposed that the main biological activity of the most potent bisphosphonate zoledronate (Zometa) in humans cells is directed against protein geranylgeranylation. This opens up the intriguing possibility that it might be possible to enhance potency by developing drugs that work by inhibiting geranylgeranyl diphosphate synthase (GGPPS, EC 2.5.1.30), the enzyme that produces the geranylgeranyl diphosphate (GGPP) used to geranylgeranylate e.g., Rac, Rap, and Rho. Based on the recent observation of a previously uncharacterized (GGPP) inhibitor site in GGPPS (4), we reasoned that larger, more hydrophobic species than those in current use might bind to this site and exhibit enhanced activity, because of increased hydrophobic stabilization and, in cells, enhanced lipophilicity. Here, we thus report structures of a series of five bisphosphonates bound to GGPPS together with, for comparative purposes, the structures of five isoprenoid diphosphate-GGPPS complexes. We find three quite different binding modes, corresponding to FPP/GPP (substrate), IPP (substrate), and GGPP [product/ inhibitor (4)...

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TcaR and IcaR are a weak and a strong negative regulator of transcription of the ica locus, respectively, and their presence prevents the poly-N-acetylglucosamine production and biofilm formation in Staphylococcus epidermidis. Although TcaR was shown to interact with the ica promoter, the precise binding region and the mechanism of interaction remained unclear. Here we present the 3D structure of TcaR in its apo form and in complex with salicylate as well as several aminoglycoside and β-lactam antibiotics. A comparison of the native and complex TcaR structures indicates that the mechanism of regulation involves a large conformational change in the DNA-binding lobe. Here, we deduced the consensus binding sequence of two [∼TTNNAA] hexamers embedded in a 16 bp sequence for a TcaR dimer. Six TcaR dimers bind specifically to three approximately 33 bp segments close to the IcaR binding region with varying affinities, and their repressor activity is directly interfered by salicylate and different classes of natural antimicrobial compounds. We also found in this study that the antimicrobial compounds we tested were shown not only to inhibit TcaR-DNA interaction but also to further induce biofilm formation in S. epidermidis in our in vivo assay. The results support a general mechanism for antibiotics in regulating TcaR-DNA interaction and thereby help understand the effect of antibiotic exposure on bacterial antibiotic resistance through biofilm formation.repressor | biofilm | DNA binding | multiple drug resistance | transcription regulation

Circular dichroism (CD) spectroscopy is an optical technique that measures the difference in the absorption of left and right circularly polarized light. This technique has been widely employed in the studies of nucleic acids structures and the use of it to monitor conformational polymorphism of DNA has grown tremendously in the past few decades. DNA may undergo conformational changes to B-form, A-form, Z-form, quadruplexes, triplexes and other structures as a result of the binding process to different compounds. Here we review the recent CD spectroscopic studies of the induction of DNA conformational changes by different ligands, which includes metal derivative complex of aureolic family drugs, actinomycin D, neomycin, cisplatin, and polyamine. It is clear that CD spectroscopy is extremely sensitive and relatively inexpensive, as compared with other techniques. These studies show that CD spectroscopy is a powerful technique to monitor DNA conformational changes resulting from drug binding and also shows its potential to be a drug-screening platform in the future.