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In vitro Potency of Novel Tetracyclines against
Pseudomonas aeruginosa and Other Major
Gram-Negative Pathogens W. O'Brien, C. Fyfe, T.
Grossman, C. Chen, R. Clark, Y. Deng, M. He, D.
Hunt, C. Sun, X. Xiao, J. Sutcliffe Tetraphase
Pharmaceuticals, Watertown, US
P 1448
Replace with logo
Contact Leland Webster Tetraphase
Pharmaceuticals lwebster_at_tphase.com
22nd ECCMID 31 March 3 April 2012 London,
United Kingdom
Objectives To discover novel tetracyclines with
enhanced Pseudomonas aeruginosa activity while
maintaining in vitro activity against other
important gram-negative pathogens. Methods
The guidances and breakpoints of the Clinical
Laboratory Standards Institute were used to
determine the susceptibility of new compounds and
comparators in microtiter-based
cation-supplemented Mueller Hinton broth or in
time-kill assays using 5 milliliter cultures. In
the case of tigecycline, FDA breakpoints (if
available) were used. In vitro potency against
Escherichia coli DH10B strains genetically
engineered to express tet(A), tet(B), tet(K),
tet(M), tet(X) or blaNDM-1 was assessed.
Compounds were also assessed for mechanism of
action (MOA) using a coupled transcription/transla
tion assay (TnT) fueled with S30 ribosomal
extracts from either E. coli or P.
aeruginosa. Results Nine novel scaffolds were
found that produced compounds with MIC90 values
of 8-16 µg/ml against recent P. aeruginosa
clinical isolates, including isolates from cystic
fibrosis (CF) patients (n96 total, including 20
CF isolates). In vitro activity against panels
of other organisms, including Acinetobacter
baumannii and extended-spectrum beta-lactamase
producing Klebsiella pneumoniae and E. coli was
also retained by many compounds, with MIC90
values of 1 µg/ml for several compounds and
comparator MIC90 values of 32 µg/ml for
tetracycline, ceftriaxone, meropenem,
levofloxacin, gentamicin, and tobramycin for P.
aeruginosa, Enterobacteriaceae and A. baumanii.
Two compounds were profiled in 24-hour time-kill
studies using 4 isolates of P. aeruginosa and
were generally found to be bactericidal at 4-8x
the MIC. The new scaffolds retained activity
against strains expressing genes encoding
tetracycline-specific efflux pumps (Tet(A),
Tet(B), Tet(K)), a ribosomal protection mechanism
(Tet(M)), and a monooxygenase that inactivates
tetracyclines (Tet(X)). The compounds inhibited
protein synthesis in both TnT assays, with IC50
values 5-10x lower than conventional
tetracyclines (1-2 µM). Conclusions This is
the first report of novel tetracyclines with
improved potency against contemporary P.
aeruginosa isolates. These compounds retain
activity against other major gram-negative
pathogens and merit additional work to advance
into development.
Activity of Compounds against Panels of Recent
Gram-negative and Gram-positive Isolates
Compound MIC50/MIC90 MIC50/MIC90 MIC50/MIC90 MIC50/MIC90 MIC50/MIC90 MIC50/MIC90 MIC50/MIC90 MIC50/MIC90 MIC50/MIC90 MIC50/MIC90 MIC50/MIC90 MIC50/MIC90
Compound (range) (range) (range) (range) (range) (range) (range) (range) (range) (range) (range) (range)
Compound Pseudomonas aeruginosa (n96) Pseudomonas aeruginosa cystic fibrosis (n20) Acinetobacter baumannii (n29) Stenotrophomonas maltophila (n15) Burkholderia cenocepacia (n10) Enterobacter cloacae (n19) Proteus mirabilis (n20) Escherichia coli ESBL (n27) Klebsiella pneumoniae ESBL (n25) Staphylococcus aureus (MRSA) (n20) Enterococcus faecalis (n21) Enterococcus faecium (n14)
TP-433 4/8 4/8 0.25/1 1/2 4/8 0.13/0.5 0.5/0.5 0.25/1 0.063/1 0.031/0.13 0.5/1 0.031/0.5
TP-433 (0.13-32) (0.13-8) (0.016 - 2) (0.25-4) (0.063-16) (0.016-2) (0.13-1) (0.016-1) (0.016-2) (0.016-1) (0.016-1) (0.016-1)
TP-559 4/8 4/8 0.25/1 0.5/1 8/32 0.25/1 1/2 0.25/1 0.25/1 0.063/0.25 1/2 0.25/1
TP-559 (0.25-32) (0.25-8) (0.016 - 2) (0.13-1) (0.13-32) (0.063-2) (0.25-2) (0.016-1) (0.063-2) (0.063-0.25) (0.031-2) (0.016-2)
TP-389 8/16 4/8 0.5/1 0.5/2 8/32 0.25/1 0.5/1 0.25/0.5 0.25/0.5 0.063/0.25 0.13/0.13 0.031/0.13
TP-389 (0.25-32) (0.25-16) (0.031-4) (0.25-2) (0.25-gt32) (0.13-1) (0.25-1) (0.063-0.5) (0.13-0.5) (0.063-0.25) (0.063-0.13) (0.016-0.13)
TP-214 8/32 8/16 2/8 4/8 32/gt32 1/8 2/4 0.5/1 1/2 0.25/0.5 2/4 0.13/2
TP-214 (0.5-32) (0.5-32) (0.25-16) (1-8) (2-gt32) (0.5-8) (1-4) (0.25-2) (0.5-2) (0.13-1) (0.25-4) (0.063-4)
TP-726 8/16 8/16 2/4 2/8 8/32 0.5/2 0.5/1 0.5/0.5 0.5/1 0.13/0.25 4/8 2/8
TP-726 (0.25-32) (0.25-16) (0.031-16) (0.5-8) (0.5-gt32) (0.25-2) (0.25-1) (0.13-1) (0.25-1) (0.13-1) (0.13-8) (0.031-16)
TP-819 8/16 8/16 0.5/1 1/2 8/32 0.25/2 2/4 0.5/1 0.25/0.5 0.13/0.25 0.25/0.25 0.063/0.25
TP-819 (0.5-32) (0.5-32) (0.031-4) (0.25-4) (0.25-gt32) (0.13-2) (2-4) (0.13-1) (0.13-1) (0.063-0.25) (0.063-0.25) (0.016-0.25)
TP-950 8/16a 4/8 1/4 1/4 8/32 0.5/2 0.5/1 2/4 0.5/4 0.13/0.25 4/8 2/8
TP-950 (0.5-gt32) (0.5-16) (0.031-8) (0.5-4) (0.5-gt32) (0.25-2) (0.25-1) (0.13-4) (0.5-8) (0.063-0.5) (0.13-8) (0.016-8)
TP-469 8/16a 8/16 2/8 4/8 16/gt32 1/4 0.5/1 4/8 1/16 0.25/0.5 4/8 0.5/8
TP-469 (0.25-32) (1-16) (0.063-32) (1-16) (1-gt32) (0.5-4) (0.25-1) (0.25-16) (0.5-gt16) (0.13-0.5) (0.25-8) (0.031-8)
TP-512 8/16b 8/16 0.5/2 0.5/1 8/32 0.5/1 2/2 0.5/0.5 0.5/1 0.13/0.25 0.25/0.25 0.063/0.25
TP-512 (1-16) (1-16) (0.016-4) (0.063-2) (0.25-gt32) (0.25-2) (1-2) (0.063-1) (0.25-2) (0.063-0.25) (0.063-0.25) (0.031-0.5)
Tetracycline gt32/gt32d ND 16/gt32 32/32 gt32/gt32 8/gt32 gt32/gt32 gt32/gt32 8/gt32 0.5/0.5 gt32/gt32 32/gt32
Tetracycline (gt32-gt32) ND (0.25-gt32) (4-gt32) (16-gt32) (2-gt32) (32-gt32) (2-gt32) (2-gt32) (0.25-32) (0.13-gt32) (0.031-gt32)
Tigecycline 16/32c ND 0.5/4 1/2 8/32 1/4 4/8 0.13/0.5 0.5/1 0.13/0.13 0.063/0.13 0.016/0.063
Tigecycline (1-gt32) ND (0.016-4) (0.25-4) (0.25-gt32) (0.25-4) (2-8) (0.063-0.5) (0.25-1) (0.063-0.25) (0.031-0.13) (0.016-0.25)
CXA-101 2/4a 1/2 32/gt32 gt32/gt32 8/32 8/gt32 1/2 gt16/gt16 gt16/gt16 gt32/gt32 gt32/gt32 gt32/gt32
CXA-101 (0.25-8) (1-8) (1-gt32) (4-gt32) (2-gt32) (0.5-gt32) (0.5-4) (0.5-gt16) (1-gt16) (gt32-gt32) (gt32-gt32) (gt32-gt32)
Tobramycin 1/gt32 1/4 ND gt32/gt32 32/gt32 ND ND ND ND ND ND ND
Tobramycin (0.13-gt32) (0.5-16) ND (2-gt32) (gt32-gt32) ND ND ND ND ND ND ND
Gentamicin ND ND 16/gt32 ND gt32/gt32 1/gt32 2/8 4/gt32 16/gt32 1/gt32 32/gt32 gt32/gt32
Gentamicin ND ND (2-gt32) ND (gt32-gt32) (0.5-gt32) (0.5-gt32) (1-gt32) (1-gt32) (0.5-gt32) (8-gt32) (32-gt32)
Meropenem 0.5/16 0.5/8 2/gt32 gt32/gt32 8/32 0.063/0.5 0.25/1 0.031/0.13 0.063/32 ND ND ND
Meropenem (0.063-gt32) (0.63-gt32) (0.13-gt32) (8-gt32) (0.5-gt32) (0.031-32) (0.063-1) (0.016-2) (0.031-gt32) ND ND ND
Levofloxacin 1/32c ND 4/32 2/8 4/8 1/16 0.13/32 16/gt32 32/gt32 8/gt32 2/gt32 gt32/gt32
Levofloxacin (0.25-gt32) ND (0.063-gt32) (0.25-8) (0.5-gt32) (0.031-gt32) (0.063-32) (0.031-gt32) (0.031-gt32) (0.25-gt32) (1-gt32) (gt32-gt32)
Ceftazidime 4/gt32c ND ND gt32/gt32 8/16 ND 0.13/0.13 32/gt32 gt32/gt32 ND ND ND
Ceftazidime (1-gt32) ND ND (4-gt32) (2-gt32) ND (0.063-0.25) (0.5-gt32) (2-gt32) ND ND ND
Ceftriaxone ND ND ND ND ND 32/gt32 ND gt32/gt32 gt32/gt32 gt32/gt32 ND gt32/gt32
Ceftriaxone ND ND ND ND ND (0.13-gt32) ND (4-gt32) (2-gt32) (gt32-gt32) ND (gt32-gt32)
Piperacillin/ Tazobactam 16/gt128c ND ND ND 16/gt128 ND ND gt128/gt128 16/gt32 ND ND ND
Piperacillin/ Tazobactam (0.5-gt128) ND ND ND (0.5-gt128) ND ND (gt128-gt128) (2/gt32) ND ND ND
Colistin 1/1 1/2 0.5/1 8/gt32 gt32/gt32 0.25/2 gt32/gt32 0.25/0.25 0.25/1 ND ND ND
Colistin (0.13-4) (0.13-4) (0.13-2) (0.13-gt32) (gt32-gt32) (0.13-gt32) (gt32-gt32) (0.13-4) (0.13-gt16) ND ND ND
Vancomycin ND ND ND ND ND ND ND ND ND 1/1 1/gt32 gt32/gt32
Vancomycin ND ND ND ND ND ND ND ND ND (0.5-1) (1-gt32) (32-gt32)
a 55 P. aeruginosa isolates (35 unbiased 20 cystic fibrosis isolates) b 20 P. aeruginosa isolates (cystic fibrosis isolates) c76 P. aeruginosa isolates (no cystic fibrosis isolated included) d35 isolates (no cystic fibrosis isolates) ND, not done a 55 P. aeruginosa isolates (35 unbiased 20 cystic fibrosis isolates) b 20 P. aeruginosa isolates (cystic fibrosis isolates) c76 P. aeruginosa isolates (no cystic fibrosis isolated included) d35 isolates (no cystic fibrosis isolates) ND, not done a 55 P. aeruginosa isolates (35 unbiased 20 cystic fibrosis isolates) b 20 P. aeruginosa isolates (cystic fibrosis isolates) c76 P. aeruginosa isolates (no cystic fibrosis isolated included) d35 isolates (no cystic fibrosis isolates) ND, not done a 55 P. aeruginosa isolates (35 unbiased 20 cystic fibrosis isolates) b 20 P. aeruginosa isolates (cystic fibrosis isolates) c76 P. aeruginosa isolates (no cystic fibrosis isolated included) d35 isolates (no cystic fibrosis isolates) ND, not done a 55 P. aeruginosa isolates (35 unbiased 20 cystic fibrosis isolates) b 20 P. aeruginosa isolates (cystic fibrosis isolates) c76 P. aeruginosa isolates (no cystic fibrosis isolated included) d35 isolates (no cystic fibrosis isolates) ND, not done a 55 P. aeruginosa isolates (35 unbiased 20 cystic fibrosis isolates) b 20 P. aeruginosa isolates (cystic fibrosis isolates) c76 P. aeruginosa isolates (no cystic fibrosis isolated included) d35 isolates (no cystic fibrosis isolates) ND, not done a 55 P. aeruginosa isolates (35 unbiased 20 cystic fibrosis isolates) b 20 P. aeruginosa isolates (cystic fibrosis isolates) c76 P. aeruginosa isolates (no cystic fibrosis isolated included) d35 isolates (no cystic fibrosis isolates) ND, not done a 55 P. aeruginosa isolates (35 unbiased 20 cystic fibrosis isolates) b 20 P. aeruginosa isolates (cystic fibrosis isolates) c76 P. aeruginosa isolates (no cystic fibrosis isolated included) d35 isolates (no cystic fibrosis isolates) ND, not done a 55 P. aeruginosa isolates (35 unbiased 20 cystic fibrosis isolates) b 20 P. aeruginosa isolates (cystic fibrosis isolates) c76 P. aeruginosa isolates (no cystic fibrosis isolated included) d35 isolates (no cystic fibrosis isolates) ND, not done a 55 P. aeruginosa isolates (35 unbiased 20 cystic fibrosis isolates) b 20 P. aeruginosa isolates (cystic fibrosis isolates) c76 P. aeruginosa isolates (no cystic fibrosis isolated included) d35 isolates (no cystic fibrosis isolates) ND, not done a 55 P. aeruginosa isolates (35 unbiased 20 cystic fibrosis isolates) b 20 P. aeruginosa isolates (cystic fibrosis isolates) c76 P. aeruginosa isolates (no cystic fibrosis isolated included) d35 isolates (no cystic fibrosis isolates) ND, not done a 55 P. aeruginosa isolates (35 unbiased 20 cystic fibrosis isolates) b 20 P. aeruginosa isolates (cystic fibrosis isolates) c76 P. aeruginosa isolates (no cystic fibrosis isolated included) d35 isolates (no cystic fibrosis isolates) ND, not done a 55 P. aeruginosa isolates (35 unbiased 20 cystic fibrosis isolates) b 20 P. aeruginosa isolates (cystic fibrosis isolates) c76 P. aeruginosa isolates (no cystic fibrosis isolated included) d35 isolates (no cystic fibrosis isolates) ND, not done
Pseudomonas aeruginosa is an old foe,
replete with 37 intrinsic multidrug-resistant
(MDR) pumps as well as metabolic pathways to
digest a variety of different substrates. It
remains a key challenge as a nosocomial pathogen,
with strains resistant to all classes of
antibiotics. Increasing resistance of P.
aeruginosa to fluoroquinolones and other
antimicrobial agents has greatly impacted
management decisions in patients with this
infection (1). Oral therapy is no longer a
treatment option in many patients. And in others,
there may be no safe and active parenterally
administered antibiotic available for use. Since
it accounts for 15-20 each of all acute
bacterial skin and skin structure infections
(ABSSSI 2) and hospital- and ventilator-associate
d pneumonia (HAP/VAP 3), and 8-10 of
complicated urinary tract infections (cUTI 4), a
drug effective against P. aeruginosa would be
life-saving. The antibacterial activity of
classic tetracycline antibiotics against P.
aeruginosa is limited by low permeability and
intrinsic efflux. Tetraphases program is aimed
at optimizing tetracyclines for anti-pseudomonal
activity. Ideally, compounds would also have
intrinsic activity against other key MDR
organisms so that the drug could be used as
empiric monotherapy to treat infections caused by
other difficult-to-treat organisms. In
exploration of fully synthetic novel scaffolds
with gram-negative activity, a number of distinct
classes were found to posses promising
anti-pseudomonal activity. This is the first
report of their antimicrobial activity against P.
aeruginosa and other key MDR pathogens.
MIC assays. Compounds were tested against panels
of unbiased sets (except where noted, i.e.
ESßL) of recent clinical isolates, including
quality control strains according to methods
published by Clinical and Laboratory Standards
Institute (CLSI) (5). Isolate collections include
strains from Eurofins Medinet (Chantilly, VA) and
IHMA (Schaumburg, IL). PCR-characterization of
extended spectrum ß-lactamases was done at IHMA
or by standard PCR methodology at Tetraphase
Pharmaceuticals using published primers to
confirm the presence of ESßL enzymes
(6). Time-kill assays. The minimal inhibitory
concentrations (MIC) were determined for
antibiotic stocks as per CLSI standardized
methodology prior to running time-kill assays.
Time-kill assays were performed as described by
CLSI guidelines (7), with the following
modifications five milliliter cultures
inoculated to a final starting density of 1 x
105 1 x 106 colony forming units (CFU) /ml
were shaken vigorously (300 rpm) at 35oC in 50 ml
polypropylene conical tubes. Cultures were
sampled at various time points, serially diluted
in sterile saline, and plated on tryptic soy
agar. The lower limit of detection per culture
was 100 CFU/ml. Antibacterial activity against
E. coli DH10B recombinantly expressing
tetracycline-resistance genes. Genes encoding
tet(A), tet(B), tet(K), tet(M), tet(X), and E.
coli ß-galactosidase (lacZ) as a control were
amplified by PCR from clinical isolates confirmed
by prior sequencing to have these
tetracycline-resistant determinants and cloned
into an L-arabinose inducible expression system
without any affinity tags (pBAD-Myc-His,
Invitrogen, Carlsbad, CA). The class B
carbapenemase NDM-1 was also cloned into the same
inducible expression system. Plasmids were
transformed and expressed in E. coli DH10B
cells (Invitrogen, Carlsbad, CA). Cloned inserts
were sequenced to verify the resistance gene
sequence and compared to reported sequences in
GenBank (accession numbers tet(A), AJ419171
tet(B), AP0961 tet(K), AJ888003 tet(M),
X90939.1 tet(X), M37699 and NDM-1, HQ162469).
Cells were grown in Mueller Hinton Broth
containing ampicillin, 50 µg/ml, pre-induced for
30 minutes with 1 arabinose (tet(A), tet(B),
tet(M), tet(X), NDM-1) or 0.3 arabinose (tet(K))
at 30?C prior to use as inocula in MIC assays
containing ampicillin, 50 ?g/ml. Susceptibility
assays were incubated at 35?C as per CLSI
guidelines. Transcription-Translation assays.
E. coli assays were run using commercially
available S30 for circular DNA (Promega Cat
L1130). P. aeruginosa extracts were prepared as
described previously (8) and were supplemented
with S30 buffer from Promega (Cat L512A) and
plasmid pBESTluc purchased from Promega (Cat.
L492A). Reactions were carried out in a total
volume of 20 µl in black-walled 96-well
flat-bottom assay plates (Costar Cat. 3915) for
one hour at 37?C. Each reaction contained 5 to
5.23 µl of extract, 0.05 to 0.6 µl of plasmid, 8
µl of S30 buffer and 3 µl of compound dilution.
Reactions were stopped by placing on ice for 5
minutes followed by the addition of 25 µl of
luciferase substrate (Promega Cat. E1500) per
well. Luminescence was read using a LUMIStar
Optima (BMG Labtech) with gain at 3600, 0.2
second read time, 0 seconds between wells.
Percent luminescence was plotted against
inhibitor concentration and the compound
concentration producing 50 inhibition (IC50) was
determined. Inhibitor IC50 values were
calculated as an average of a minimum of three
independent IC50 determinations.
Compound Inhibition of In Vitro
Transcription/Translation in E. coli and P.
aeruginosa S30 Extracts
In Vitro Activity against Recombinantly Expressed
Resistance Mechanisms in E. coli DH10B
Compound MIC (µg/ml) MIC (µg/ml) MIC (µg/ml) MIC (µg/ml) MIC (µg/ml) MIC (µg/ml) MIC (µg/ml)
Compound E. coli DH10B strain expressing E. coli DH10B strain expressing E. coli DH10B strain expressing E. coli DH10B strain expressing E. coli DH10B strain expressing E. coli DH10B strain expressing E. coli DH10B strain expressing
Compound lacZ tet(M) tet(K) tet(A) tet(B) tet(X) NDM-1
TP-433 0.0312 0.5 0.0312 4 2 4 0.016
TP-559 0.0625 0.5 0.0312 4 0.125 0.5 0.0625
TP-389 0.25 0.5 0.125 1 0.5 1 0.25
TP-214 0.5 4 0.5 1 1 4 0.25
TP-726 0.25 4 0.125 1 0.5 1 0.25
TP-819 0.125 0.25 0.125 4 0.25 1 0.125
TP-950 0.25 4 0.0625 16 0.5 1 0.25
TP-469 0.25 2 0.125 32 1 2 0.5
TP-512 0.25 0.25 0.25 1 0.25 1 ND
Tetracycline 4 128 128 gt128 gt128 128 ND
Ceftriaxone 0.125 0.125 0.0625 0.125 0.0625 0.125 gt32
Compound P. aeruginosa P. aeruginosa E. coli E. coli
Compound IC50 (µg/ml) SD IC50 (µg/ml) SD
TP-433 0.12 0.04 0.25 0.08
TP-559 0.11 0.03 0.24 0.05
TP-389 0.25 0.13 0.26 0.05
TP-214 0.23 0.09 0.37 0.02
TP-726 0.25 0.07 0.36 0.08
TP-819 0.27 0.12 0.37 0.05
TP-950 0.30 0.11 0.65 0.06
TP-469 0.25 0.01 0.60 0.00
TP-512 0.21 0.03 0.33 0.04
Tetracycline 1.40 0.86 1.87 0.21
Linezolid 0.94 0.39 1.53 0.06
TP-433 and TP-559 are Bactericidal In Vitro

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• The Tetraphase synthetic chemistry platform has
  enabled the synthesis of multiple distinct
  classes of novel tetracyclines with potent,
  mechanism-based in vitro activity against P.
  aeruginosa which translates to in vivo efficacy
  (see posters P1425 and P1426).
• Several of these lead classes show broad-spectrum
  potency against panels of multidrug-resistant
  organisms and bactericidal activity against P.
  aeruginosa.
• This is the first demonstration of novel
  tetracyclines with potential for use against P.
  aeruginosa and other difficult-to-treat
  Gram-negative and Gram-positive infections.