Dr Lynne Silver

1. Why novel antibacterial discovery is
so hard and what to do about it
SWON Industry Workshop
September 22, 2016
Lynn L. Silver
LL Silver Consulting, LLC

2. The “Innovation gap”in novel classes
Obscures 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

3. Antibacterials at FDA 2000-2015
Compound Usage Class Active versus
resistance
Discovery 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

4. 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

5. Inhibit bacterial growth
Small molecule ‘Leads’Small molecule ‘Leads’
Small molecule ‘Hits’Small molecule ‘Hits’
since the mid-90s
Gene-to-Drug Approach
Novel antibacterial targets
High Throughput Screening
Candidates
Genomics
Preclinical testing
Clinical Trials
Drug
Inhibit the enzyme
Inhibit bacterial growth by
inhibiting the enzyme
Druglike properties
Low resistance potential
ez
abez ab
Candidates

6. 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

7. Examine successful antibacterials to
get a handle on resistance potential

8. Systemic Monotherapies

9. Single-Enzyme Targeted Drugs
Silver, L. L. (2016). Cold Spring Harbor perspectives in medicine:a030239.

10. Based on existing antibacterial drugs…
• Successful monotherapeutic antibacterials
– Not subject to single-step mutation to high level resistance
because 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.

11. 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

12. “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
Hernandez, V.,et al.. 2013. Antimicrob. Agents Chemother. 57:1394-1403.
Twynholm, M., et al. 2013. Poster -1251 at 53rd ICAAC, Denver
O'Dwyer, K., A. Spivak, et al. (2014). Antimicrob. Agents Chemother. epub

13. 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.

14. Antibacterial Multitargeting
GlcNAc
MurNAc PP-C55
Gyrase Topo IV
Lipid II
ciprofloxacin
daptomycin
vancomycin
gentamicin
tetracycline
chloramphenicol
linezolid
erythromycin
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…

15. Cytoplasmic Entry:
How drugs get into
Gram-negative cells

16. -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
Periplasm
Cytoplasm
P. aeruginosa
Spectrum is due to permeability & efflux
Spectrum

17. 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.

18. G- barriers to G+ agents
S. Aureus
MIC
E. 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 OM
Novobiocin 0.05 200 50 0.8 0.4 612 1.4/3.3
fold wt 4 250 500 Efflux
Erythromycin 0.25 250 3.9 1.0 0.25 732 2.9/3.9
fold wt 64 250 1000 Efflux & OM
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

19. 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

20. 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

21. Antibacterials Are Chemically Unlike other Drugs
Gram-negative
Gram-positive only
Other drugs +
MW
cLogD7.4
O'Shea, R. O. and H. E. Moser (2008). J. Med. Chem. 51: 2871-2878.

22. 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
( )

23. 92 Cytoplasm-targeted registered antibacterials
Silver, L. L. (2008). Exp. Opin. Drug Disc. 3(5): 487-500

24. -12
-10
-8
-6
-4
-2
0
2
4
6
8
0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400
Gram-negative
GN Transported & AG
Gram-positive only
MW
CLogD7.4
Do we need more bins?
Silver, L. L. (2016) A Gestalt approach to
Gram-negative entry. Bioorg. Med. Chem.
131 compounds

25. 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

26. 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]
ψ
aminoglycosidesfosfomycin
chloramphenicolalbomycin

27. 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

29. GN compounds entering by diffusion

30. GP compounds in GN space,
but effluxed

31. Transported compounds that might be
able to diffuse
MW ClogD7.4
bacilysin 270 -4.49
negamycin 248 -5.87
streptozotocin 265 -1.45
nojirimycin 179 -2.37
D-cycloserine 102 -1.85
fosfomycin 138 -5.99

32. Erythromycin Azithromycin L-701,677 Cmpd 15
MIC μg/ml
S. pneumoniae 0.02 0.03 0.03 ≤0.06
S. aureus 0.25 1 0.5 1
E. faecalis 1 4 2 2
E. coli 32 1 1 0.13
H. influenzae 2 0.5 1 0.25
K. pneumoniae 32 2 1 0.13

33. 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
rifamycin
trimethoprim
vancomycin
novobiocin
cycloserine
lincomycin
cephalosporin
chlortetracycline
chloramphenicol
streptomycin
bacitracin
penicillin
sulfonamide
metronidazole
erythromycin
isoniazid
Last novel agent to reach the
clinic was discovered in 1984
pleuromutilin
2010
DaptomycinLinezolid
Bactroban
Synercid
Retapamulin
Norfloxacin
Imipenem
cephamycin
lipiarmycin
Fidaxomicin
Modification of old classes
has proceeded – but no newly
discovered novel classes have
been registered at FDA in 32 years