why novel antibacterial discovery is so hard
TRANSCRIPT
Why novel antibacterial discovery is so hard and what to do about it
SWON Industry WorkshopSeptember 22, 2016
Lynn L. Silver LL Silver Consulting, LLC
The “Innovation gap”in novel classesObscures the “Discovery void”
Fischbach and Walsh, 2009
Oxazolidinones
Glycopeptides
Macrolides
Aminoglycosides
Chloramphenicol, Tetracyclines - lactams
Mutilins
Sulfa drugs
Innovation gap
No registered classes of antibiotics were discovered after 1984
Between 1962 and 2000, no major classes of antibiotics were introduced
Discovery void
Lipopeptides
1950 1960 1980 1990 2000 20101940 1970
Quinolones, Streptogramins
Antibacterials at FDA 2000-2015Compound Usage Class Active versus
resistanceDiscovery of class
Fail at FDA
Pass at FDA
Linezolid Systemic IV/oral Oxazolidinones MRSA 1978 2000
Ertapenem Systemic IV/IM Carbapenem 1976 2001
Cefditoren Systemic oral Cephalosporin 1948 2001
Gemifloxacin Systemic oral Fluoroquinolone 1961 2003
Daptomycin Systemic oral Lipopeptide MRSA 1984 2003
Telithromycin Systemic oral Macrolide+ EryR S. pneumo 1952 2004
Tigecycline Systemic IV Tetracycline+ TetR 1948 2005
Faropenem Systemic oral Penem 1978 2006
Retapamulin Topical Pleuromutilin MRSA 1952 2007
Dalbavancin Systemic IV Glycopeptide 1953 2007 2014
Doripenem Systemic IV Carbapenem 1976 2007
Oritavancin Systemic IV Glycopeptide+ VRE 1953 2008 2014
Cethromycin Systemic oral Macrolide+ EryR S. pneumo 1952 2009
Iclaprim Systemic IV Trimethoprim+ TrmR 1961 2009
Besifloxacin Ophthalmic Fluoroquinolone 1961 2009
Telavancin Systemic IV Glycopeptide+ VRE 1953 2009
Ceftobiprole Systemic IV Cephalosporin+ MRSA 1948 2009
Ceftaroline Systemic IV Cephalosporin+ MRSA 1948 2010
Fidaxomicin Oral CDAD Lipiarmycin 1975
Tedizolid Systemic IV/Oral Oxazolidinone 1978 2014
Avy-Caz Systemic IV Cephalosporin+BLI CRE 1948+ 2015
Ceftolozane Systemic IV Cephalosporin+BLI 1948 2014
Consider…• If Big Pharma (and biotechs) have been largely
unsuccessful in finding novel antibacterials to develop…
• Will that be reversed by– Increasing financial incentives?– Revising regulatory policy?
• What has prevented novel discovery?• The need to address scientific obstacles
Inhibit bacterial growthSmall molecule ‘Leads’Small molecule ‘Leads’
Small molecule ‘Hits’Small molecule ‘Hits’
since the mid-90s
Gene-to-Drug ApproachNovel antibacterial targets
High Throughput Screening
Candidates
Genomics
Preclinical testing
Clinical Trials
Drug
Inhibit the enzyme
Inhibit bacterial growth by inhibiting the enzyme
Druglike propertiesLow resistance potential
Compounds kill by other means
Same as for other drugs
Almost all have high resistance potential
ezabez ab Candidates
Compounds can’t enter
Why has it been so hard?• Opportunity cost
– Too much time chasing “targets”– Not enough time addressing rate limiting steps
• Rate limiting steps– Defining resistance potential of targets– Chemistry
• Getting things into cells & avoiding efflux• Better chemical libraries / return to natural products
Examine successful antibacterials to get a handle on resistance potential
Systemic Monotherapies
Single-Enzyme Targeted Drugs
Silver, L. L. (2016). Cold Spring Harbor perspectives in medicine:a030239.
Based on existing antibacterial drugs…• Successful monotherapeutic antibacterials
– Not subject to single-step mutation to high level resistancebecause they are multi-targeted
• Current drugs inhibiting single enzymes – Generally used in combination
because they are subject to single mutation to significant resistance
THUS: "Multitargets" are preferable to single enzyme targets for systemic monotherapy
BUT: The search for single enzyme inhibitors has been the mainstay of novel discovery for at least 20 years …
Silver, L. L. and Bostian, K. A. (1993). Antimicrob. Agents. Chemother. 37:377-83.; Silver, L. L. (2007). Nat. Rev. Drug Discov. 6:41-55.
If single enzyme targets give rise to resistance in the laboratory…
• Determine if the in vitro (laboratory) resistance is likely to translate to resistance in the clinic– Standardize the use of models for evolution of resistance under
therapeutic conditions • Hollow fiber system in vitro• Animal models with high inoculum
– Is “overnight” resistance likely to occur?
• Develop fixed combinations– To prevent resistance as in TB, HIV, HCV, etc.
• Pursue multitargets
“Overnight” resistance GSK’052 (AN3365)
• Oxaborole inhibitor of Leucyl tRNA Synthetase• Excellent Gram-negative spectrum• In vitro resistance frequencies of >10-8
• In Phase 2b cUTI study, resistance occurred in 4 of 14 patients post treatment (3 after one day of treatment)
• Mutants were highly fit and MICs raised >1000 fold• This should have been predictable
O
B
NH2
OHOHO
Hernandez, V.,et al.. 2013. Antimicrob. Agents Chemother. 57:1394-1403.Twynholm, M., et al. 2013. Poster -1251 at 53rd ICAAC, DenverO'Dwyer, K., A. Spivak, et al. (2014). Antimicrob. Agents Chemother. epub
Hollow fiber (in vitro) resistance study of GSK’052• GSK’052 dosed vs E. coli at high (108/ml) inocula• Resistant mutants take over the population in one day
VanScoy, B. D., et al. 2013. Poster A-016 at 53rd ICAAC, Denver.
Antibacterial Multitargeting
GlcNAc
MurNAc PP-C55
Gyrase Topo IV
Lipid II
ciprofloxacin
daptomycinvancomycin
gentamicintetracyclinechloramphenicollinezoliderythromycin
Target the products of multiple genes – or the product of their function – such that single mutations cannot lead to high level resistance• Two or more essential gene products with
similar active sites: DNA Gyrase & Topisomerase IV• Products of identical genes : rRNA• Essential structures produced by a pathway where
structural changes cannot be made by single mutations: Membranes
• These and other known multiargets have been pursued • But no new multitargeted agents have reached the clinic…
Cytoplasmic Entry:How drugs get into Gram-negative cells
-lactamsGlycopeptides
CycloserineFosfomycin
Rifampin
AminoglycosidesTetracyclines
ChloramphenicolMacrolides
LincosamidesOxazolidinones
Fusidic AcidMupirocin
NovobiocinFluoroquinolones
SulfasTrimethoprimMetronidazole
DaptomycinPolymyxin
Gram-positive
CM
Cytoplasm
OM
Gram-negative
CMPe
ripla
sm
Cytoplasm
P. aeruginosa
Spectrum is due to permeability & efflux
Spectrum
But the spectrum may mislead
• Since the major permeability difference between Gram- and Gram+ is the OM, some assume that finding ways of transiting the OM and avoiding efflux will allow Gram- entry
• This is an error based on the fact that OM-permeable and effluxΔ Gram-negatives are sensitive to many Gram-positive drugs.
G- barriers to G+ agentsS. Aureus
MICE. coli MIC (g/mL) Major barrier MW ClogD7.4 / ClogP
wt lpxC tolC lpxC tolC
Rifampicin 0.0008 5 0.005 2.5 0.005 823 2.8/3.6 fold wt 1000 2 1000 OMNovobiocin 0.05 200 50 0.8 0.4 612 1.4/3.3 fold wt 4 250 500 EffluxErythromycin 0.25 250 3.9 1.0 0.25 732 2.9/3.9 fold wt 64 250 1000 Efflux & OM
O
NH
OH
OOOO
O
OH2N
O
OH
OH
NHOH
O
OHOH
O OHO
O
O
O
HOO
NN
N
O
O
O
O
OOH
HO
N
OO
OOH
OH
Kodali S, Galgoci A, Young K et al. J. Biol. Chem. 280(2), 1669-1677 (2005)
These G+ agents already have properties that allow them to cross the cytoplasmic membrane
However, If you start with random inhibitors and endow them with qualities allowing OM-passage and efflux-avoidance they are unlikely to enter the cytoplasm
The physicochemical characteristics for OM passage and efflux avoidance appear orthogonal to those for CM passage
Gram negative barriers• The Outer Membrane (OM) of gram negatives adds an orthogonal
barrier to that of the cytoplasmic membrane
Penetration of 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 You could study the selectivity of the barriers, transporters, porins, pumps individually OR – you could ask what kind of molecules can enter the gram negative cytoplasm?
OM
CM
periplasm
A Gestalt approach to Gram-negative entry
• Turn from characterizing barriers individually• To characterizing compounds that can enter• Can we develop rules for entry by studying existing
compounds?• In 2008, O’Shea and Moser published the first
analysis of physicochemical characteristics of registered antibacterials making the distinction between G- and G+ actives
Antibacterials Are Chemically Unlike other Drugs
Gram-negative
Gram-positive only
Other drugs +
MW
cLog
D 7.4
O'Shea, R. O. and H. E. Moser (2008). J. Med. Chem. 51: 2871-2878.
Binning Antibacterials
O'Shea, R. O. and H. E. Moser (2008]Silver, L. L. (2011). Clin. Microbiol. Rev. 24(1): 71-109 based on data from O’Shea and Moser)
0.50.5
( )
92 Cytoplasm-targeted registered antibacterials
Silver, L. L. (2008). Exp. Opin. Drug Disc. 3(5): 487-500
0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400-12
-10
-8
-6
-4
-2
0
2
4
6
8
Gram-negative
GN Transported & AG
Gram-positive only
MW
CLog
D 7.4
Do we need more bins?
Silver, L. L. (2016) A Gestalt approach toGram-negative entry. Bioorg. Med. Chem.
131 compounds
Can we bin by route of entry?
• Measure entry of (thousands of)compounds into the cytoplasm (independent of activity)
• Determine routes of entry through OM, efflux potential, CM.
• Determine a set of physico-chemical and/or structural parameters (rules) for each bin
Routes to the cytoplasm
OM
CM
periplasm
LPS &O-Ag
• Diffusion– Hydrophilic molecules: Cross OM rapidly via porins, may avoid efflux –poor CM passage– Lipophilic molecules: Cross OM slowly, can be effluxed – good CM passage
• Active– Hydrophilic molecules cross OM via porins, CM via transporters [ATP or PMF driven]
• Self-promoted uptake [SPU] through OM– Cationic molecules, avoid efflux, CM passage via ψ or anionic lipid sequestration– Watch for toxicity!
• Trojan horse– Piggyback on active or facilitated transport; must avoid rapid resistance
• OM permeabilizers and EPIs as adjuncts– Combine with CM-transiting molecules [properties of Gram+ drugs]
ψaminoglycosidesfosfomycin
chloramphenicolalbomycin
Antibacterial Discovery is a Multipronged Problem
• Rational drug discovery focuses on structural biology of targets– But single targets are resistance-prone– Can we use combinations? Multitargets?
• For Gram-negative antibacterials, must also study physicochemistry of entry, LPS structure, efflux.– Can we devise rules based on routes of entry?
• Multiple parameters must be optimized simultaneously for successful drug design
• Produce “Gram-negative” chemical libraries– Screen more empirically
N
N
O
O
O
NH2
H2N
CH3
CH3H3C
HN
Cl
ClOH
O2N HOO
N
OHF
N
O O
HN
ciprofloxacin
chloramphenicol
trimethoprim
N
N
OH
O2N
CH3
SNH
NO
CH3
OO
H2N
metronidazole
sulfamethoxazole
ON N NH
O
O
O2N
nitrofurantoin
NH
HN
O
OOHH3C
N
O
OH
CHIR-090
O
OH Cl
ClCl
triclosan
OH
NH2
OOHOH OO
NCH3HO
OH
CH3
tetracycline HH
GN compounds entering by diffusion
clindamycin
O
O
O
OHO
H3C CH3 HO
OH
CH3 O
CH3
HO
CH3
CH3
O
CH3
HO
CH3
O
OH
O
H3C
H3C
fusidic acid
mupirocin
N
O
N
OO
HN CH3
O
F
linezolid
nargenicin A1
NH
N
N
CH3OO
OCH3
Debio 1452
platensimycin
OH
O
NH
OO
O
OH
CH3OOH3C
CH3
OH
CH3
CH3
O
H
H
H
HH
H
H
HH
H
NH
O
OCH3
OOH
OH
H3C
OH
O
H
H
ONH
N
H3C
CH3 O
OH
HO OH
S
CH3
H
CH3Cl
H
H
H
H
GP compounds in GN space, but effluxed
Transported compounds that might be able to diffuse
NHO
H2N O
Nh2
HN
NOH
OH Nh2 O OHH
CH3
negamycinD-cycloserine OH
O
NH
O
NH2
H3C
O
O
bacilysinO
HO
OH
OH
NH
NCH3
NO
OHO
streptozotocin
HN OH
OHHO
OH
HO
nojirimycin
MW ClogD7.4bacilysin 270 -4.49negamycin 248 -5.87streptozotocin 265 -1.45nojirimycin 179 -2.37D-cycloserine 102 -1.85 fosfomycin 138 -5.99
O
CH3OH
HO
O
fosfomycin
Erythromycin Azithromycin L-701,677 Cmpd 15
MIC μg/mlS. pneumoniae 0.02 0.03 0.03 ≤0.06S. aureus 0.25 1 0.5 1E. faecalis 1 4 2 2E. coli 32 1 1 0.13H. influenzae 2 0.5 1 0.25K. pneumoniae 32 2 1 0.13
O
O
O
HOHOHO
O
O
O
OOH
NHO
ON
O
O
O
HOHOHO
O
O
O
OOH
NHO
O
N
O
O
HOHOHO
O
O
O
OOH
NHO
O
N
O
O
HOHOHO
O
O
O
ONH2
NHO
Discovery Timeline
1935
1940
1945
1955
1950
1965
1960
1970
1975
1980
1985
1990
1995
2000
2005
1930
fusidic acid
polymyxin
oxazolidinones
daptomycin
carbapenem
monobactams
mupirocin
fosfomycin
streptogramins
nalidixic acid
rifamycintrimethoprim
vancomycin
novobiocincycloserine
lincomycin
cephalosporin
chlortetracyclinechloramphenicol
streptomycin
bacitracin
penicillinsulfonamide
metronidazole
erythromycinisoniazid
Last novel agent to reach the clinic was discovered in 1984
pleuromutilin
2010
DaptomycinLinezolid
Bactroban Synercid
Retapamulin
NorfloxacinImipenem
cephamycinlipiarmycin
Fidaxomicin
Modification of old classeshas proceeded – but no newly discovered novel classes havebeen registered at FDA in 32 years