Transcript Slide 1
P 1448 22
nd
ECCMID 31 March – 3 April 2012 London, United Kingdom 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
Contact: Leland Webster Tetraphase Pharmaceuticals [email protected]
Revised Abstrac
t
Objectives:
To discover novel tetracyclines with enhanced while maintaining
in vitro Pseudomonas aeruginosa
activity against other important gram-negative pathogens.
activity
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. I
n vitro
potency against
Escherichia coli
DH10B strains genetically engineered to express
tet
(A),
tet
(B),
tet
(K),
tet
(M),
tet
(X) or
bla
NDM-1 was assessed. Compounds were also assessed for mechanism of action (MOA) using a coupled transcription/translation assay (TnT) fueled with S30 ribosomal extracts from either
aeruginosa.
E. coli
or
P.
Results:
Nine novel scaffolds were found that produced compounds with MIC 90 μg/ml against recent
P. aeruginosa
values of 8-16 clinical isolates, including isolates from cystic fibrosis (CF) patients (n=96 total, including 20 CF isolates).
In vitro
activity against panels of other organisms, including
Acinetobacter baumannii
and extended-spectrum beta-lactamase producing meropenem, levofloxacin, gentamicin, and tobramycin for
Klebsiella pneumoniae
and
E. coli
was also retained by many compounds, with MIC 90 several compounds and comparator MIC 90 values of values of ≤1 μg/ml for ≥32 μg/ml for tetracycline, ceftriaxone,
P. aeruginosa, Enterobacteriaceae
and
A. baumanii
.
aeruginosa
Two compounds were profiled in 24-hour time-kill studies using 4 isolates of and were generally found to be bactericidal at 4-8x the MIC.
P.
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 IC 50 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.
Results Activity of Compounds against Panels of Recent Gram-negative and Gram-positive Isolates
MIC 50 /MIC 90 (range) Compound
Pseudomonas aeruginosa
(n=96)
Pseudomonas aeruginosa
(n=20)
Acinetobacter Stenotrophomona baumannii
(n=29) (n=15)
Burkholderia
cystic fibrosis (n=10)
Enterobacter
(n=19)
Proteus Escherichia cloacae
(n=20)
coli ESBL
(n=27)
Klebsiella pneumoniae
(n=25)
Staphylococcus
(n=20)
Enterococcus
(n=21)
Enterococcus
(n=14) TP-433 TP-559 TP-389 TP-214 TP-726 TP-819 TP-950 TP-469 TP-512 Tetracycline Tigecycline CXA-101 Tobramycin Gentamicin Meropenem
4/8 (0.13-32) 4/8 (0.25-32) 8/16 (0.25-32) 8/32 (0.5-32) 8/16 (0.25-32) 8/16 (0.5-32) 8/16 a (0.5->32) 8/16 a (0.25-32) 8/16 b (1-16) >32/>32 d (>32->32) 16/32 c (1->32) 2/4 a (0.25-8) 1/>32 (0.13->32) ND 0.5/16 (0.063->32) 1/32 c 4/8 (0.13-8) 4/8 (0.25-8) 4/8 (0.25-16) 8/16 (0.5-32) 8/16 (0.25-16) 8/16 (0.5-32) 4/8 (0.5-16) 8/16 (1-16) 8/16 (1-16) ND ND 1/2 (1-8) 1/4 (0.5-16) ND 0.5/8 (0.63->32) 0.25/1 (≤0.016 - 2) 0.25/1 (≤0.016 - 2) 0.5/1 (0.031-4) 2/8 (0.25-16) 2/4 (0.031-16) 0.5/1 (0.031-4) 1/4 (0.031-8) 2/8 (0.063-32) 0.5/2 (0.016-4) 16/>32 (0.25->32) 0.5/4 (≤0.016-4) 1/2 (0.25-4) 0.5/1 (0.13-1) 0.5/2 (0.25-2) 4/8 (1-8) 2/8 (0.5-8) 1/2 (0.25-4) 1/4 (0.5-4) 4/8 (1-16) 0.5/1 (0.063-2) 32/32 (4->32) 1/2 (0.25-4) 4/8 (0.063-16) 8/32 (0.13-32) 8/32 (0.25->32) 32/>32 (2->32) 8/32 (0.5->32) 8/32 (0.25->32) 8/32 (0.5->32) 16/>32 (1->32) 8/32 (0.25->32) >32/>32 (16->32) 8/32 (0.25->32) 0.13/0.5
(≤0.016-2) 0.25/1 (0.063-2) 0.25/1 (0.13-1) 1/8 (0.5-8) 0.5/2 (0.25-2) 0.25/2 (0.13-2) 0.5/2 (0.25-2) 1/4 (0.5-4) 0.5/1 (0.25-2) 8/>32 (2->32) 1/4 (0.25-4) 0.5/0.5
(0.13-1) 1/2 (0.25-2) 0.5/1 (0.25-1) 2/4 (1-4) 0.5/1 (0.25-1) 2/4 (2-4) 0.5/1 (0.25-1) 0.5/1 (0.25-1) 2/2 (1-2) >32/>32 (32->32) 4/8 (2-8) 0.25/1 (≤0.016-1) 0.25/1 (≤0.016-1) 0.25/0.5
(0.063-0.5) 0.5/1 (0.25-2) 0.5/0.5
(0.13-1) 0.5/1 (0.13-1) 2/4 (0.13-4) 4/8 (0.25-16) 0.5/0.5
(0.063-1) >32/>32 (2->32) 0.13/0.5
(0.063-0.5) 0.063/1 (≤0.016-2) 0.25/1 (0.063-2) 0.25/0.5
(0.13-0.5) 1/2 (0.5-2) 0.5/1 (0.25-1) 0.25/0.5
(0.13-1) 0.5/4 (0.5-8) 1/16 (0.5->16) 0.5/1 (0.25-2) 8/>32 (2->32) 0.5/1 (0.25-1) 0.031/0.13
(≤0.016-1) 0.063/0.25
(0.063-0.25) 0.063/0.25
(0.063-0.25) 0.25/0.5
(0.13-1) 0.13/0.25
(0.13-1) 0.13/0.25
(0.063-0.25) 0.13/0.25
(0.063-0.5) 0.25/0.5
(0.13-0.5) 0.13/0.25
(0.063-0.25) 0.5/0.5
(0.25-32) 0.13/0.13
(0.063-0.25) 0.5/1 (≤0.016-1) 1/2 (0.031-2) 0.13/0.13
(0.063-0.13) 2/4 (0.25-4) 4/8 (0.13-8) 0.25/0.25
(0.063-0.25) 4/8 (0.13-8) 4/8 (0.25-8) 0.25/0.25
(0.063-0.25) >32/>32 (0.13->32) 0.063/0.13
(0.031-0.13) 0.031/0.5
(≤0.016-1) 0.25/1 (≤0.016-2) 0.031/0.13
(≤0.016-0.13) 0.13/2 (0.063-4) 2/8 (0.031-16) 0.063/0.25
(≤0.016-0.25) 2/8 (≤0.016-8) 0.5/8 (0.031-8) 0.063/0.25
(0.031-0.5) 32/>32 (0.031->32) ≤0.016/0.063
(≤0.016-0.25) 32/>32 (1->32) ND 16/>32 (2->32) 2/>32 (0.13->32) >32/>32 (4->32) >32/>32 (2->32) ND >32/>32 (8->32) 8/32 (2->32) 32/>32 (>32->32) >32/>32 (>32->32) 8/32 (0.5->32) 8/>32 (0.5->32) ND 1/>32 (0.5->32) 0.063/0.5
(0.031-32) 1/2 (0.5-4) ND 2/8 (0.5->32) 0.25/1 (0.063-1) >16/>16 (0.5->16) ND 4/>32 (1->32) 0.031/0.13
(≤0.016-2) >16/>16 (1->16) ND 16/>32 (1->32) 0.063/32 (0.031->32) >32/>32 (>32->32) ND 1/>32 (0.5->32) ND >32/>32 (>32->32) ND 32/>32 (8->32) ND >32/>32 (>32->32) ND >32/>32 (32->32) ND 4/32 2/8 (0.25-8) 4/8 (0.5->32) 1/16 0.13/32 16/>32 (0.031->32) (0.063-32) (0.031->32) 32/>32 (0.031->32) 8/>32 (0.25->32) 2/>32 (1->32) >32/>32 (>32->32)
Introduction
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-associated 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 low permeability and intrinsic efflux.
P. aeruginosa
is limited by Tetraphase’s 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 MDR pathogens.
P. aeruginosa
and other key
Methods
MIC assays.
Compounds were tested against panels of unbiased sets (except where noted, i.e.
“ESβL + ”) 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 β-lactamases was done at IHMA or by standard PCR methodology at Tetraphase Pharmaceuticals using published primers to confirm the presence of ES βL 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 10 5 – 1 x 10 6 colony forming units (CFU) /ml were shaken vigorously (300 rpm) at 35 o C 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
resistance genes.
Genes encoding
tet
DH10B recombinantly expressing
(A),
tet(
B),
tet(
K),
tet(
M),
tet
(X), and
tetracycline-
E. coli
β 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
tet
(M), μg/ml, pre-induced for 30 minutes with 1% arabinose (
tet
(A),
tet
(B),
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 pBEST
luc
of 20 purchased from Promega (Cat. # L492A). Reactions were carried out in a total volume 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 (IC 50 ) was determined. Inhibitor IC 50 were calculated as an average of a minimum of three independent IC 50 determinations.
values
References 1.
2.
3.
4.
5.
6.
7.
8.
Sader, HS, R. Mallick, A. Kuznik, T.R. Fritsche, and R.N. Jones.
2007. Use of
in vitro
susceptibility and pathogen prevalence data to model the expected clinical success rates of tigecycline and other commonly used antimicrobials for empirical treatment of complicated skin and skin-structure infections. Int. J. Antimicro.b Agents 30:514-520. Epub 2007 Oct 23.
Itani, K.M.F., S. Merchant, S.-J. Lin, K. Akhras, J.C. Alandete, and H.T. Hatoum.
2011. Outcomes and management costs in pastients hospitalized for skin and skin-structure infections. Am. J. Infect. Contro.l 39:42-49.
Jones, R.N.
2010. Microbial etiologies of hospital-acquired bacterial pneumonia and ventilator-associated bacterial pneumonia. Clin. Infect. Di.s 51 Suppl 1:S81-87.
Wagenlehner, F.M.E., W. Weidner, G. Perletti, and K. G. Naber.
2010. Emerging drugs for bacterial urinary tract infedtions. Expert Opin. Emerging Drugs 15:375-397.
Clinical and Laboratory Standards Institute (CLSI).
2009. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard —Eighth Edition. CLSI document M07-A8 [ISBN 1-56238-689-1]. Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA.
Dallenne, C., A. Da Costa, D. Decre, C. Favier, and G. Arlet.
2010. Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in
Enterobacteriaceae.
J. Antimicrob. Chemother. 65:490-495.
CLSI.
1999. Methods for determining bactericidal activity of antimicrobial agents; approved guideline. CLSI document M26-A., vol. 19, CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania, USA.
Fyfe, C., T. Farrell, J. Sutcliffe, and T. Grossman.
2011. Abstr. 51 st Annual Intersci. Conf. Antimicrob. Agents Chemother,. abstr. F2-158. Development and characterization of a coupled transcription/translation (TNT) assay from
P. aeruginosa
.
In Vitro Activity against Recombinantly Expressed Resistance Mechanisms in E. coli DH10B
Compound TP-433 TP-559 TP-389 TP-214 TP-726 TP-819 TP-950 TP-469 TP-512 Tetracycline Ceftriaxone
lacZ
0.0312
0.0625
0.25
0.5
0.25
0.125
0.25
0.25
0.25
4 0.125
MIC (µg/ml) tet(M) E. coli DH10B strain expressing: tet(K) tet(A) tet(B) tet(X)
0.5
0.5
0.0312
0.0312
4 4 2 0.125
4 0.5
0.5
4 0.125
0.5
1 1 0.5
1 1 4 4 0.25
4 2 0.25
128 0.125
0.125
0.125
0.0625
0.125
0.25
128 0.0625
1 4 16 32 1 >128 0.125
0.5
0.25
0.5
1 0.25
>128 0.0625
1 1 1 2 1 128 0.125
NDM-1
≤0.016
0.0625
0.25
0.25
0.25
0.125
0.25
0.5
ND ND >32
Compound Inhibition of In Vitro Transcription/Translation in E. coli and P. aeruginosa S30 Extracts
Compound TP-433 TP-559 TP-389 TP-214 TP-726 TP-819 TP-950 TP-469 TP-512 Tetracycline Linezolid
P. aeruginosa
IC 50 (µg/ml)
0.12
0.11
0.25
0.23
0.25
0.27
0.30
0.25
0.21
1.40
0.94
SD
0.04
0.03
0.13
0.09
0.07
0.12
0.11
0.01
0.03
0.86
0.39
E. coli
IC 50 (µg/ml)
0.25
0.24
0.26
0.37
0.36
0.37
0.65
0.60
0.33
1.87
1.53
SD
0.08
0.05
0.05
0.02
0.08
0.05
0.06
0.00
0.04
0.21
0.06
TP-433 and TP-559 are Bactericidal In Vitro Conclusions
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.