Post on 10-Mar-2020
Why is it so hard to develop antibacterial drugs for Gram negative
bacteria?
Lynn L. Silver, Ph.D.
LL Silver Consulting, LLC
Why is it so hard to discover antibacterial drugs for Gram negative
bacteria?
2
The “Innovation gap”in novel classes
Obscures the “Discovery void”
Fischbach and Walsh, 2009
Oxazolidinones
Glycopeptides
Macrolides
Aminoglycosides
Chloramphenicol, Tetracyclines
Quinolones, Streptogramins
- lactams
Mutilins
Sulfa drugs
Innovation gap
No registered classes of antibiotics were discovered after 1987
Between 1962 and 2000, no major classes of antibiotics were introduced
Discovery void
Lipopeptides
1950 1960 1980 1990 2000 2010 1940 1970
3
Failure to find new antibacterials during this period…
Due largely to reliance on using “novel” targets to screen/design in vitro inhibitors without paying attention to the obstacles to further development
1. Inhibitors of single enzyme targets are likely to select for rapid resistance [in the lab]
2. Compounds discovered active in vitro may will have a hard time entering bacteria
1. Much more problematic in Gram-negatives
2. Compound libraries not compatible with bacterial entry
4
-lactams Glycopeptides
Cycloserine Fosfomycin
Rifampin
Aminoglycosides
Tetracyclines Chloramphenicol
Macrolides Lincosamides
Oxazolidinones Fusidic Acid Mupirocin
Novobiocin
Fluoroquinolones Sulfas
Trimethoprim Metronidazole
Daptomycin Polymyxin
Gram-positive
CM
Cytoplasm
OM
Gram-negative
CM Pe
rip
lasm
Cytoplasm
P. aeruginosa
For Gram-negatives, limiting factors are entry and efflux
5
Gross Structural Differences
If the target is in the Gram-negative cytoplasm, then the drug must traverse 2 membranes and avoid efflux
If the target is in the periplasm [as are the PBPs, targets of β-lactams], then the drug needs “only” to traverse the outer membrane and avoid efflux
Gram Negative Bacteria Ga
peptidoglycan cell wall
peptidoglycan cell wall
Gram Positive Bacteria
cytoplasm cytoplasm
Target Location is Key
6
Diffusion through a phospholipid bilayer like the cytoplasmic membrane (CM) requires uncharged, lipophilic species
Polar, hydrophilic, charged compounds require active
transport
Cytoplasmic membrane (CM) barrier
ADP+Pi ATP
CM
However, active transport permeases have not been found for most marketed antibacterials
7
Gram negative barriers
The Outer Membrane (OM) of gram negatives adds an orthogonal barrier to that of the cytoplasmic membrane
ATP
Penetration of the OM – through porins – prefers small (<600 MW) hydrophilic, charged compounds
But highly charged molecules can’t penetrate the CM (unless actively transported)
Molecules that do penetrate can be effluxed from the cytoplasm – or periplasm
What kind of molecules can enter the gram negative cytoplasm?
OM
CM
periplasm
8
-6.0
-4.0
-2.0
0.0
2.0
4.0
6.0
8.0
0 200 400 600 800 1000 1200
MW
cLo
gP
Gram positive only Cytoplasmic
Gram negative cytoplasmic entry by diffusion
Cytoplasmic energy dependent transport
LL Silver 2008. Exp. Opin. Drug Disc. 3:487-500
91 compounds
Cytoplasm-targeted antibacterials
SNH
NO
OO
H2N
OH
NH2
OOHOH OO
NOH
OH
N
N
O
O
O
NH2
H2N
N
OHF
N
O O
HN
chloramphenicol tetracycline ciprofloxacin
metronidazole sulfamethoxazole nitrofurantoin
N
N
OH
O2N
Cl
ClOH
O2N HOO
ON N NH
O
O
O2N
trimethoprim
9
Are there rules for G- entry by diffusion?
Can a set of rules be arrived at With sufficient data from many more chemotypes
Measurement of entry not dependent on activity
Chemical descriptors cLogD at pH 6.5 through 8 MW pKa / charge Radius PSA etc
cLogD7.4 -1 to +2 MW < 500 Charge pH 7.5 -1 to 0
10
But some compounds use “tricks”
Eg: To cross the outer membrane, cations (or polycations) can use “self-promoted uptake”
Locally disrupt the outer membrane to enter the periplasm
Efflux will still play a role
Crossing the cytoplasmic membrane may be PMF-dependent (ΔΨ)
Proposed by Bob Hancock in 1984
11
Mg++
Mg++
Mg++
EDTA chelates Mg++, disrupts LPS
EDTA at 1 mM
Lipid A LPS
12
Compounds proposed to cross the OM by Self-promoted uptake
Polymyxin B
Aminoglycosides [tobramycin]
Deglucoteicoplanin-polyamine
Azithromycin
Merck amino-azalide
Merck IMP-inhibitor
Trius GyrB/ParE?
Rib-X 04 series? At pH 7.4 all are dibasic or more
13
Self-promoted uptake by bivalent molecules?
Mg++ Mg++
Rib-X 04
Local disruption at ≤ μM concentrations
Mg++
14
Most important for P. aeruginosa and other lactose non-fermenters but also for enterics
Structural information and computer modeling predicts two rather promiscuous binding sites.
Will it be possible to find broad spectrum inhibitors?
Or design compounds to avoid efflux?
Efflux
15
Multipronged Problem
Rational drug discovery focuses on structural biology of targets
For Gram-negative antibacterials, must also study structural biology/chemistry of entry, LPS structure, efflux.
But, there is limited structural knowledge for approaching these barriers rationally
Multiple parameters must be optimized simultaneously for successful drug design
16
For anti-Gram negative agents
Need robust understanding of entry and efflux requirements
There are new Gram-negative agents in the pipeline Rib-X protein synthesis inhibitor
Trius dual topoisomerase inhibitor
Achaogen LpxC inhibitor [no structure released yet]
Improvements in existing classes to specifically overcome pre-existing resistance can and have been made Achaogen - Aminoglycosides
Tetraphase - Tetracyclines
New β-lactam / β-lactamase inhibitor combinations
For all these new agents, entry and efflux as well as target inhibition, were tracked throughout optimization