Pseudomonas aeruginosa strains are less susceptible to tigecycline (previously GAR-936; MIC, 8 g/ml) than many other bacteria (P. J. Petersen, N. V. Jacobus, W. J. Weiss, P. E. Sum, and R. T. Testa, Antimicrob. Agents Chemother. 43:738-744, 1999). To elucidate the mechanism of resistance to tigecycline, P. aeruginosa PAO1 strains defective in the MexAB-OprM and/or MexXY (OprM) efflux pumps were tested for susceptibility to tigecycline. Increased susceptibility to tigecycline (MIC, 0.5 to 1 g/ml) was specifically associated with loss of MexXY. Transcription of mexX and mexY was also responsive to exposure of cells to tigecycline. To test for the emergence of compensatory efflux pumps in the absence of MexXY-OprM, mutants lacking MexXY-OprM were plated on medium containing tigecycline at 4 or 6 g/ml. Resistant mutants were readily recovered, and these also had decreased susceptibility to several other antibiotics, suggesting efflux pump recruitment. One representative carbenicillin-resistant strain overexpressed OprM, the outer membrane channel component of the MexAB-OprM efflux pump. The mexAB-oprM repressor gene, mexR, from this strain contained a 15-bp in-frame deletion. Two representative chloramphenicol-resistant strains showed expression of an outer membrane protein slightly larger than OprM. The mexCD-OprJ repressor gene, nfxB, from these mutants contained a 327-bp in-frame deletion and an IS element insertion, respectively. Together, these data indicated drug efflux mediated by MexCD-OprJ. The MICs of the narrower-spectrum semisynthetic tetracyclines doxycycline and minocycline increased more substantially than did those of tigecycline and other glycylcyclines against the MexAB-OprM-and MexCD-OprJ-overexpressing mutant strains. This suggests that glycylcyclines, although they are subject to efflux from P. aeruginosa, are generally inferior substrates for P. aeruginosa efflux pumps than are narrower-spectrum tetracyclines.Pseudomonas aeruginosa is a clinically important gram-negative opportunistic pathogen causing serious acute and chronic infections (8, 11). The exceptional array of intrinsic and acquired drug resistance mechanisms employed by P. aeruginosa renders antibiotic treatment of these infections problematic. One important resistance mechanism is efflux mediated by the so-called resistance nodulation division (RND) family of efflux pumps. Four RND pumps have been described in P. aeruginosa: MexAB-OprM (16), MexCD-OprJ (31), , and 25,40). RND pumps consist of an inner membrane transporter (MexB, MexD, MexF, and MexY), an outer membrane channel-forming component (OprM, OprJ, and OprN), and a membrane fusion protein (MexA, MexC, MexE, and MexX) (27). RND pumps show broad specificity, and their tripartite architecture allows extrusion of compounds directly from the cytoplasm to the external environment. Efflux pump action in P. aeruginosa leads to particularly high levels of drug resistance as a result of apparent synergism with the atypically impermeable outer membrane, which limits influx of antimic...

The 9-t-butylglycylamido derivative of minocycline (TBG-MINO) is a recently synthesized member of a novel group of antibiotics, the glycylcyclines. This new derivative, like the first glycylcyclines, theN,N-dimethylglycylamido derivative of minocycline and 6-demethyl-6-deoxytetracycline, possesses activity against bacterial isolates containing the two major determinants responsible for tetracycline resistance: ribosomal protection and active efflux. The in vitro activities of TBG-MINO and the comparative agents were evaluated against strains with characterized tetracycline resistance as well as a spectrum of recent clinical aerobic and anaerobic gram-positive and gram-negative bacteria. TBG-MINO, with an MIC range of 0.25 to 0.5 μg/ml, showed good activity against strains expressing tet(M) (ribosomal protection), tet(A), tet(B),tet(C), tet(D), and tet(K) (efflux resistance determinants). TBG-MINO exhibited similar activity against methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant streptococci, and vancomycin-resistant enterococci (MICs at which 90% of strains are inhibited, ≤0.5 μg/ml). TBG-MINO exhibited activity against a wide diversity of gram-negative aerobic and anaerobic bacteria, most of which were less susceptible to tetracycline and minocycline. The in vivo protective effects of TBG-MINO were examined against acute lethal infections in mice caused by Escherichia coli, S. aureus, andStreptococcus pneumoniae isolates. TBG-MINO, administered intravenously, demonstrated efficacy against infections caused byS. aureus including MRSA strains and strains containingtet(K) or tet(M) resistance determinants (median effective doses [ED50s], 0.79 to 2.3 mg/kg of body weight). TBG-MINO demonstrated efficacy against infections caused by tetracycline-sensitive E. coli strains as well asE. coli strains containing either tet(M) or the efflux determinant tet(A), tet(B), ortet(C) (ED50s, 1.5 to 3.5 mg/kg). Overall, TBG-MINO shows antibacterial activity against a wide spectrum of gram-positive and gram-negative aerobic and anaerobic bacteria including strains resistant to other chemotherapeutic agents. The in vivo protective effects, especially against infections caused by resistant bacteria, corresponded with the in vitro activity of TBG-MINO.

Aggrecanases are now believed to be the principal proteinases responsible for aggrecan degradation in osteoarthritis. Given their potential as a drug target, we solved crystal structures of the two most active human aggrecanase isoforms, ADAMTS4 and ADAMTS5, each in complex with bound inhibitor and one wherein the enzyme is in apo form. These structures show that the unliganded and inhibitor-bound enzymes exhibit two essentially different catalytic-site configurations: an autoinhibited, nonbinding, closed form and an open, binding form. On this basis, we propose that mature aggrecanases exist as an ensemble of at least two isomers, only one of which is proteolytically active.Keywords: protein structure; enzymes; metalloproteins; aggrecanases Supplemental material: see www.proteinscience.org Osteoarthritis (OA) is a progressive disease that results in degradation of articular cartilage and chronic pain. The extracellular matrix is composed of two major components, aggrecan and collagen. Aggrecan is a large multidomain proteoglycan that provides cartilage with compressibility and elasticity by swelling and hydrating the collagen network (Vertel and Ratcliffe 2000). Loss of aggrecan is considered a critical early event in OA, occurring initially at the joint surface and progressing to the deeper zones. This is followed by degradation of collagen fibrils and mechanical failure of the tissue (Nagase and Kashiwagi 2003). Aggrecanase-1 (ADAMTS4) and aggrecanase-2 (ADAMTS5), members of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) gene family, cleave aggrecan at a unique site termed the ''aggrecanase site Tortorella et al. 1999). ADAMTS4 and ADAMTS5 are expressed in human normal and OA cartilage (Yamanishi et al. 2002) and in OA synovium, and contribute to the structural damage that characterizes human OA (Powell et al. 2007;Song et al. 2007). However, there is no consensus in the literature as to which aggrecanase is the most important in human OA. In mice, ADAMTS5 (but not ADAMTS4) is responsible for disease progression in a surgically induced model of OA (Glasson et al. 2004(Glasson et al. , 2005. ADAMTS4/ADAMTS5 double knockout mice are physiologically normal (Majumdar et al. 2007) and also protected from developing OA. Given the normal phenotype of the double knockout mice, dual inhibition Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

Tigecycline (GAR-936) and daptomycin are potent antibacterial compounds in advanced stages of clinical trials. These novel agents target multiply resistant pathogenic bacteria. Daptomycin is principally active against gram-positive bacteria, while tigecycline has broad-spectrum activity. When tested by the standard protocols of the National Committee for Clinical Laboratory Standards in Mueller-Hinton broth II, tigecycline was more active than daptomycin (MICs at which 90% of isolates tested are inhibited, 0.12 to 1 and 0.5 to 16 g/ml, respectively) against staphylococcal, enterococcal, and streptococcal pathogens. Tigecycline (GAR-936), a glycylcycline (36), and daptomycin, a lipopeptide (1), are novel antibacterial compounds undergoing clinical development. Tigecycline is a broad-spectrum, protein-inhibiting, antibacterial agent possessing activity against strains resistant to other chemotherapeutic agents (14,29). Daptomycin, a cell wall-inhibiting antibiotic with a spectrum of activity limited to gram-positive bacteria, has also been demonstrated to have activity against resistant bacteria (34). Early clinical trials with daptomycin were discontinued due to less-than-desired outcomes (32) including unwanted side effects on skeletal muscle. However, new dosage regimens (27) have allowed daptomycin to progress into clinical trials (37). These antibacterial agents offer new alternatives for the treatment of infections caused by clinically relevant pathogens for which limited therapeutic options exist.The rise in the incidence of methicillin-resistant Staphylococcus aureus (MRSA) strains (28) and the emergence of strains with intermediate glycopeptide resistance (38) have emphasized the lack of therapeutic alternatives. Recently, a collection of glycopeptide-intermediate S. aureus (GISA) strains with reduced susceptibilities to the glycopeptide antibiotics (vancomycin and teicoplanin) has been assembled by the Network on Antibiotic Resistance in Staphylococcus aureus (NARSA). That study was undertaken to evaluate the in vitro activities of tigecycline, daptomycin, and comparative antibiotics against these GISA and other drug-resistant gram-positive isolates by the standard methodology of the National Committee for Clinical Laboratory Standards (NCCLS) (26). The activity of daptomycin was determined in both Mueller-Hinton broth II (MHB II) and Mueller-Hinton broth supplemented with 50 mg of calcium per liter. In addition, the effects of calcium concentration and the culture medium on the activities of the antibiotics were determined for the GISA, MRSA, and methicillin-susceptible S. aureus (MSSA) isolates, as daptomycin is a calcium-dependent antibiotic. The supplemental calcium concentrations (25, 50, and 75 mg/liter) recommended by other investigators (34) were used for these studies. MATERIALS AND METHODSOrganisms. Routine clinical isolates were collected from various medical centers in the United States and Canada between 1990 and 1999. Identification of each culture was performed by conventional methodologie...

We examined the influence of substituents in tetracycline (tc) analogs modified at positions 2 and 4-9 and anhydrotetracycline (atc) on induction of the Tn10-encoded Tet repressor (TetR) by a quantitative in vitro induction assay. The equilibrium association constants of the modified tc to TetR were independently determined to distinguish effects on binding from those on induction. We found a correlation between the binding affinity and induction of TetR for most tc analogs. While a substitution at position 5 revealed only minor effects, changes at position 6 increased binding and induction efficiencies up to 20-fold. A chlorine at position 7 or 8 enhanced binding and induction about 4- and 9-fold, respectively. Substituents at position 9 decreased binding up to 5-fold. Epimerization of the dimethylamino function at position 4 in 4-epi-tc resulted in about 300-fold-reduced binding and 80-fold-reduced induction. Substitution of this grouping by hydrogen in 4-de(dimethylamino)-tc resulted in no binding and no induction. The respective atc analog failed to induce as well, although binding was still observed. The dimethylamino function may, thus, play a role in triggering the conformational change of TetR necessary for induction. Substitution of the 2-carboxamido by a nitrilo function did not influence binding and induction efficiencies. Atc showed about 30-fold increased binding and induction, being the most effective inducer tested in this study. The equilibrium association constants of most TetR-[Mg-tc]+ and TetR-([Mg-tc]+)2 analog complexes with tet operator are decreased about 10(2)- and 10(8)-fold, respectively, as compared to those of free TetR. This suggests that these tc analogs share the same molecular mechanism of TetR induction.

N,N-Dimethylglycylamido (DMG) derivatives of minocycline and 6-demethyl-6-deoxytetracycline are new semisynthetic tetracyclines referred to as the-glycylcyclines. The in vitro activities of the glycylcyclines were evaluated in comparison with those of minocycline and tetracycline against strains carrying characterized tetracycline resistance determinants and against 995 recent clinical isolates obtained from geographically distinct medical centers in North America. The glycylcyclines were active against tetracycline-resistant strains carrying efflux [tet(A), tet(B), tet(C), and tet(D) in Escherichia coli and tet(K) in Staphylococcus aureus] and ribosomal protection [tet(M) in S. aureus, Enterococcusfaecalis, and E. coli)] resistance determinants. Potent activity (MIC for 90% of strains, c0.5 ,ug/ml) was obtained with the glycylcyclines against methicillinsusceptible and methicillin-resistant S. aureus, E. faecalis, Enterococcus faecium, and various streptococcal species. The glycylcyclines exhibited good activity against a wide diversity of gram-negative aerobic and anaerobic bacteria, most of which were less susceptible to minocycline and tetracycline. The activities of the glycylcyclines against most organisms tested were comparable to each other. The in vivo efficacies of the glycylcyclines against acute lethal infections in mice when dosed intravenously were reflective of their in vitro activities. The glycylclines had efficacies comparable to that of minocycline against infections with methicillin-susceptible and methicillin-resistant S. aureus strains, a strain carrying tet(K), and a tetracyclinesusceptible E. coli strain but exceeded the effectiveness of minocycline against infections with resistant isolates, including strains harboring tet(M) or tet(B). Levels of DMG-6-demethyl-6-deoxytetracycline in serum were higher and more sustained than those of DMG-minocycline or minocycline. Our results show that the glycylcyclines have potent in vitro activities against a wide spectrum of gram-positive and gram-negative, aerobic and anaerobic bacteria, including many resistant strains. On the basis of their in vitro and in vivo activities, the glycylcyclines represent a significant advance to the tetracycline class of antibiotics and have good potential value for clinical efficacy.The tetracyclines, first isolated at Lederle Laboratories in 1945 from a strain of Streptomyces aureofaciens, represented a significant advance in the treatment of many infections (11). The activity of the tetracyclines against a wide variety of gram-positive and gram-negative aerobic and anaerobic bacteria, mycoplasmas, and rickettsiae and their efficacy against both intracellular and extracellular pathogens permitted their widespread use (12). Through modifications of the fermentation conditions and semisynthetic synthesis, several analogs, such as minocycline (MINO) and doxycycline, which exhibited improved antimicrobial activity and more favorable pharmacokinetic properties over those of the early tetracyclines were prepared (20, 27...

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This report describes the discovery of a new generation of tetracycline antibacterial agents, the "glycylcyclines". These agents are notable for their activity against a broad spectrum of tetracycline-susceptible and -resistant Gram-negative and Gram-positive aerobic and anaerobic bacteria possessing various classes of tetracycline-resistant determinants [tet B (efflux), tet M (ribosomal protection)]. The design and synthesis of a number of 7-substituted 9-substituted-amido 6-demethyl-6-deoxytetracyclines are described.