Transcript Eight Antifungal Classes

Recent Advances in
Antifungal Drug Development
Jennifer O’Neill
February 2, 2006
1
Outline


History
Marketed Drug Classes





Polyenes
Azoles
Echinocandins
Future Targets
Conclusions
2
Dramatic Increase

300% as many hospital-acquired
fungal infections


Increase in immunocompromised
population (HIV/AIDS)
Changes in medical practice




Immunosuppressive drugs
Harsh chemotherapy
Indwelling catheters
Indiscriminate use of broad
spectrum antibiotics
Current Treatment Options in Infectious Diseases 2003, 5, 489.
Images from web.princeton.edu and www.sai1.net
3
Types of Fungal Infections

Candidiasis – Candida albicans




Impaired immunity, receiving broad-spectrum
antibiotic treatment
80% of hospital-acquired infections
Mortality rate ~ 40%
Aspergillosis – Aspergillus spp.



Impaired immunity, corticosteroid recipients
1/3 infected – never received antifungal therapy
Mortality rate ~ 80%
de Pauw, B. E.; Meuier F. Chemotherapy 1999, 45, 1.
Images from DoctorFungus Corporation
4
Impact of Infections
Heart transplant
patients die of
invasive
aspergillosis
Lung transplant
patients die of
invasive
aspergillosis
21%
Infectionrelated deaths
in leukemia
patients
35%
de Pauw, B. E.; Meunier F. Chemotherapy 1999, 45, 1.
Image from DoctorFungus Corporation
HIV/AIDS
patients will
contract fungal
infections
25%
90%
5
Fungi Challenging to Target
Archaea
KINGDOMS
Fungi
Filamentous
Yeasts
Eukaryotes
Animals
Bacteria

Cellular similarities



Complicates target identification
Diversity of structure
Diversity of metabolic targets
Image from kvhs.nbed.nb.ca
6
Too Few Antifungals


Genetic tools unavailable
Down-played for many decades

Far fewer infections (until 1980s)

Inhibitory cost

200 patents from 1998–2000

10–12 years to clinic
7
Necessary Characteristics





Target resistant species
Wide therapeutic window
Minimal host toxicity
Minimal drug-drug interactions
Exhibit in vivo fungicidal, not
fungistatic activity
Current Treatment Options in Infectious Diseases 2003, 5, 489.
8
Antifungal Classes








Polyenes
bind ergosterol
Azoles
inhibit ergosterol synthesis
Echinocandins
inhibit glucan synthase
Allylamines
inhibit squalene epoxidase
Nikkomycins
chitin synthesis inhibitors
Sodarins
inhibit protein synthesis
N-Myristoyl transferase inhibitors
Sphingolipid synthesis inhibitors
9
OH
OH
O
CH3
HO
O
OH OH
OH
OH OH HO
O
OH
H3C
O
O
HO
Polyenes
H2N
OH
Binding ergosterol
10
Key Events in Polyene History
1940s
1950s
1949
First polyene
identified:
Nystatin
1960s
1970s
1980s
1990s
2000s
1960
Amphotericin B
approved
1956
Amphotericin B
activity reported
Sheehan, D. J. et al. Clin. Microbiol. Rev. 1999, 12(1), 40
1990-92
Lipid formulations
of Amphotericin B
introduced
11
Amphotericin B

Isolated from bacteria in
1956

Streptomyces noursei
OH
HO
OH
O
H2N
HO
O
O
OH
OH
HO
HO

The gold standard



Most effective antifungal
for over three decades
Fungicidal
Limited to fungi that
contain sterols
OH
HO
HO
O
O
CH3
H3C
OH
12
Mechanism of Action

OH

Amphotericin B
binds to ergosterol
in cell membrane
Alters permeability
of membrane
NH2
OH
HO
ergosterol
Amphotericin B
O
H3C
H
HO
H
O
O
HO
O
OH OH
CH3
O
OH
Ghannoum, M. A.;Rice L. B. Clin. Microbiol. Rev. 1999, 12(4), 501.
Milhaud, J. et al. Biochim. Biophys. Acta 2002, 1558, 95.
OH OH HO
OH
OH
OH
13
Mechanism of Action
OH
aggregates
Ghannoum, M. A.; Rice L. B. Clin. Microbiol. Rev. 1999, 12(4), 501.
Milhaud, J. et al. Biochim. Biophys. Acta 2002, 1558, 95.
Aqueous pores
cause leakage of
vital cytoplasmic
components
14
Limitations of Amphotericin B

Drug of last resort – highly toxic
FUNGAL
H
MAMMALIAN
vs.
H
HO
H
HO
Ergosterol

H
Cholesterol
Resistance has been reported

Fungi alter membrane composition
15
R'
N
OR
X
N
N
X
Azoles
Blocking ergosterol synthesis
16
Key Events in Azole History
1940s
1950s
1944
First antifungal
azole reported
1958
First azole antifungal
marketed:
Ketoconazole
1990s
2000s
1990-92
Fluconazole &
Itraconazole
introduced
2002
Voriconazole
(Pfizer)
approved
1993-95
Second generation
triazoles reported
2005
Posaconazole
(Schering)
approved
Sheehan, D. J. et al. Clin. Microbiol. Rev. 1999, 12(1), 40
17
R'
Mechanism of Action
N
OR
X
N
N
X
azoles
HO
Lanosterol


HO
Inhibits cytochrome P450
14a-demethylase
Fungistatic, not fungicidal
Ghannoum, M. A.; Rice L. B. Clin. Microbiol. Rev. 1999, 12(4), 501.
Image from Podust, L. M. et al. PNAS 2001, 98(6), 3068.
18
1st Generation Triazoles


Major impact on management of fungal
infections in 1990s
Broad spectrum of activity


Yeasts and filamentous fungi
1999: >15 marketed azoles worldwide
N
N
N
N
OH
F
N
N
F
O
N
Fluconazole
O
N
N
N
N
O
N
N
O
Cl
Cl
N
Itraconazole
19
N
N
N
N
OH
F
N
N
Rate of Infection*
Fluconazole
9
8
7
6
5
4
3
2
1
0
F


91
93
Year
95
97
100
(%)
High safety profile –
extensive use
Not active against
Aspergillus spp.
Increasing reports of
antifungal resistance
89
Proportion

C. albicans
non-albicans
80
60
40
20
0
92
93
94
95
96
Year
97
98
99
*blood stream infections/
10,000 central venous catheter days
Ghannoum, M. A.; Rice L. B. Clin. Microbiol. Rev. 1999, 12(4), 501.
Trick, W. E. et al. Clin. Infect. Dis. 2002, 35, 627.
Hope, W. et al. J. Hosp. Infect. 2002, 50, 56.
20
2nd Generation Triazoles




Enhanced potency (10–500x) over 1st generation
Broad-spectrum activity: yeasts, molds, Aspergillus
Excellent central nervous system penetration
Greatly reduced toxicity
N
F
N
N
O
O
N
N
N
OH
F
Voriconazole
N
N
N
N
N
N
O
F
F
N
HO
Posaconazole
F
Koltin Y.; Hitchcock C.A. Curr. Opin. Chem. Biol. 1997, 1(2), 176.
Groll A. H.; Walsh, T. J. Swiss Med. Wkly. 2002, 132, 303.
21
Derivatives of Fluconazole
N
Wanted to increase
spectrum of activity to
include Aspergillus spp.
N
F
N
OH X
Y
R3
R1
F
R2
R1 = H, Me
R2 = H, F, Cl
R3 = H, Cl
X =N, CH
Y = N, CH
Synthesis of fluoropyrimidine
MeONa
O
F
O
OC2H5
H2N
HN
NH
F
H
POCl3
N
O
reflux
H2, Pd/C
N
N
F
Cl
Dickinson R. F. et al. Bioorg. Med. Chem. Lett. 1996, 6(16), 2031.
EtOH, 20 °C
N
F
N
22
In vitro Activity of Azoles
N
N
N
N
F
N
OH
F
N
N
N
N
N
F
Fluconazole (Flu)
O
N
N
N
Voriconazole (Vor)
N
N
O
Cl
(mg/mL)*
Flu
Itr
Vor
Aspergillus
fumigatus
>50
0.39
0.09
Candida
albicans
1.00
0.12
0.03
Candida
krusei
>25
0.05
0.24
Candida
glabrata
1.90
0.19
0.19
9.6
0.39
0.39
OH
F
F
O
MIC
N
N
O
Itraconazole (Itr)
N
N
Cryptococus
neoformans
Cl
*minimum inhibitory concentration
Dickinson R. F. et al. Bioorg. Med. Chem. Lett. 1996, 6(16), 2031.
23
N
Voriconazole
F
N
N




N
N
a-CH3 gives a marked increase in
activity
Pyrimidine ring expands therapeutic
window
Side effects
Multiple drug-drug interactions
Dickinson R. F. et al. Bioorg. Med. Chem. Lett. 1996, 6(16), 2031.
Ghannoum, M. A.; Rice L. B. Clin. Microbiol. Rev. 1999, 12(4), 501.
OH
F
F
24
Drug-Drug Interactions
Rifampin
Efavirenz
Rifabutin
Barbiturates
Phenytoin
Terfenadine
HIV Protease Inhibitors
Astemizole
NNRTIs
Sirolimus
Cisapride
Pimozide
Quinidine
Ergot Alkaloids
Cyclosporine
Methadone
Tacrolimus
Warfarin
Omeprazole
Benzodiazepine
Vinca Alkaloids
HMG-CoA Reductase Inhibitors
Sulfonylurea Oral Hypoglycemics
Dihydropyridine Calcium Channel Blockers
Pfizer Inc. VFEND® Complete Product Information, March 2005.
25
Quantitative SAR Study



No 3-D structural data available in Candida
Homology and pharmacophore modeling
5 structure classes: A–E
N
N
MeO
N
Cl
A
N
R
N
Cl
B
R
N
N
N
N
R
N
N
N
R1
R
C
N
R1
D
Di Santo R. et al. J. Med. Chem. 2005, 48, 5140
N
2
R
E
26
Synthesis of Class A
Cl
Ts
NaOH
O
Cl
EtOH
Cl
O
O
C N
NaH
DMSO, Et2O
Cl
Cl
R-I, K2CO3
Cl
N
H
CHO
O
O
Cl
LiAlH4
Cl
N
R
HO
THF
Cl
N
N
N
N
Cl
N
R
Di Santo R. et al. J. Med. Chem. 2005, 48, 5140
DMF
N
N
MeCN
Cl
Cl
N
R
27
In Vitro Anti-Candida Activity

Tested in 12 Candida albicans strains
N
N
MeO
Cl
A
N
Cl
B
N
R
MIC = 0.74–3.9 mg/mL
R
N
N
N
N
3.5–340 mg/mL
24 mg/mL
N
R
N
D
Fluconazole
0.24 mg/mL
C
R1
R
N
R1
N
N
2.5–26 mg/mL
Di Santo R. et al. J. Med. Chem. 2005, 48, 5140
E
N
2
R
0.07–220 mg/mL
28
Pharmacophore Generation



Training set: Classes A–E
activities spanned 4 orders
of magnitude (n=24, r2=0.93)
Whole set (n = 64, r2 = 0.73)
The most active compounds
matched all pharmacophore
features


All from Class E
Fluconazole matched 3 of 4
UNA = unsubstituted Ar N EV = excluded volumes
HY = hydrophobic
RA = aromatic ring
Di Santo R. et al. J. Med. Chem. 2005, 48, 5140
29
Activity Prediction
N
Cl
N
N
R
Class E
Cmpd
X
Expt
Calc
Error
1
CH3
0.025
0.13
5.1
2
C3H7
0.023
0.0064
-3.6
3
CH2-C3H5
0.025
0.052
2.1
4
CH=CH2
0.031
0.26
8.3
5
CH2CH=CH2
0.019
0.0076
-2.5
6
CH2CH=(CH3)2
0.043
0.063
1.5
0.069
0.59
8.6
Flu
N
N
N
N
Values expressed
as MICcmpd/MICbif
OH
F
N
N
F
fluconazole
N
N
Calc/
Expt
bifonazole
Di Santo R. et al. J. Med. Chem. 2005, 48, 5140
30
Azole Summary





2nd generation targets resistant strains
Broad spectrum activity
Far less toxic than amphotericin B
Multiple drug-drug interactions
Fungistatic
31
HO
HO
H3C
O
HO
O
NH
N
HO
O
OH
NH
O
OH
O HN
NH O
H
N
N
OH
O
OH
Echinocandins
HO
Targeting the fungal cell wall
32
Key Events for Echinocandins
1940s
1950s
1960s
1990s
1988
First
echinocandin
tested
Sheehan, D. J. et al. Clin. Microbiol. Rev. 1999, 12(1), 40
2000s
2001
Caspofungin
(Merck)
approved
33
Mechanism of Action

Non-competitive inhibitors of b(1,3)-glucan synthase
O
OH
O
HO
HO
OH
O
OH
O P O P O
OH O
O
HO
N
O
+
HO
HO
O
OH
HO
O
O
O
OH
HO
HO
OH
OH
OH
OH
OH
NH
O
HO
O HO
O
O
OH
OH
O
O
OH
OH
Mannoproteins
Cell
wall
b(1,6)-glucan
b(1,3)-glucan
Chitin
Phospholipid bilayer
of cell membrane
b(1,3)glucan synthase
Image from DoctorFungus Corporation
Sawistowska-Schroder E. T. et al. FEBS Lett. 1984, 173(1), 134.
34
HO
Echinocandins
HO
H3C
HO
O
NH
N
HO
O
OH
O
NH
O
HN
OH
O
NH O
H
N
N
OH
O
OH

Fungicidal




HO
Causes rapid lysis in growing cells
Candida & Pneumocystis carinii activity
Fewer drug-drug interactions
Three in clinical development:

Caspofungin, micafungin, anidulafungin
Letscher-Bru, V.; Herbrecht R. J. Antimicrob. Chemother. 2003, 51, 513.
35
SAR of Simplified Analogs
HO
HO
H3C
HO
NH
R
O
OH
O HN
NH O
H
N
O
O
NH
N
HO
O
OH
O
O
NH
N
simplify
HO
O
N
OH
O
HO
NH
R
O
OH
O HN
NH O
H
N
N
O
OH
HO

R=
O(CH2)7CH3
Replaced unusual amino acids

L-homotyrosine crucial for antifungal activity

L-threonine could replace 3-hydroxy-4-methyl proline
Zambias R. A. et al. J. Med. Chem. 1993, 35, 2843
36
Sidechain SAR Study
HO
HO
H3C
O
HO
O
NH
N
HO
O
OH
NH R
O
OH
O HN
NH O
H
N
R = -(CH2)n-CH3
n=11–21
OR'
R’ = -(CH2)n-CH3
n=5–13
NH
R’ = -(CH2)n-CH3
n=6–15
R'
N
O(CH2)7CH3
O(CH2)7CH3
(cilofungin)
(o, m, p)
OH
O
OH
O(CH2)4CH3
O
HO



Too long: hemolytic in vitro
Too short: no antifungal activity
C log P > 3.5 = antifungal
Debono J. et al. J. Med. Chem. 1995, 38, 3271
37
Cationic Derivatives
HO
HO


Cilofungin withdrawn
due to toxicity of
solubilizing agent
Increase water solubility
Unique regio-, chemo-,
and stereoselective
synthesis from core


O
NH
H
H2N

OH
O
NH
O
HN
OH
O
N
O
NH O
H
N
HO
O
HO
N
OH
O
OH
Pneumocandin B
HO
H3N
O
HO
H
H3N
4 linear steps
83% yield
HO
O
NH
N
HO
O
OH
O
NH
O
HN
OH
O
NH O
H
N
OC7H15
N
OH
O
OH
HO
Bouffard, F. A. et al. J. Med. Chem. 1994, 37, 222.
Journet, M. et al. J. Org. Chem. 1999, 64, 2411.
38
Pneumocandin Semi-Synthesis
 Pneumocandin Bo isolated from Glarea lozoyensis
 Most efficient route began with acylation of amine
HO
HO
O
HO
O
HO
HO
O
HO
NH
N
H2N
OH
O
NH
O
HN
OH
O
NH O
H
N
N
1. enzymatic hydrolysis
C6F5O2C
OH
2.
O
OH
HO
Journet, M. et al. J. Org. Chem. 1999, 64, 2411.
O
HO
O
, TEA
HO
OC7H15
O
O
NH
N
H2N
OH
NH
O
HN
OH
O
NH O
H
N
OC7H15
N
OH
O
98%
OH
HO
39
Dehydration and Etherification
 Direct reduction of amide gave mixture of products
 Protection of benzylic alcohol required
HO
HO
O
O
HO
O
HO
NH R
O
OH
O HN
NH O
H
N
N
OH
HO
1. cyanuric chloride
DMF/H2O, -30 °C
2. PhB(OH)2
3.
OH
O
HO
O
O
NH
N
H2N
CbzHN
OH
CbzHN
HO
O
OH
CCl3CO2H
HO
4. H2O
92%
(99:1 a/b)
O
NH
N
NC
OH
O
NH R
O
HN
OH
O
NH O
H
N
N
OH
O
OH
HO
R=
OC7H15
Journet, M. et al. J. Org. Chem. 1999, 64, 2411.
40
One Pot Hydrogenation
 Hydrogenation of nitrile
 Deprotection of Cbz-protected amine
CbzHN
H2N
O
HO
HO
O
HO
O
O
NH
N
NC
OH
O
NH R
O
OH
O HN
NH O
H
N
N
HO
5 mol % Pd/Al2O3
10 mol % Rh/Al2O3
H2N
H2 (40 psi), 25 °C
35 eq NH4OAc
5% HOAc
HO
OH
O
OH
HO
O
NH
N
HO
O
OH
O
NH R
O
OH
O HN
NH O
H
N
N
OH
O
92%
OH
HO
R=
OC7H15
Journet, M. et al. J. Org. Chem. 1999, 64, 2411.
41
H2N
Caspofungin
NH
HO
H2N



N
HO
NH
O
OH
O HN
NH O
H
N
N
OH
O
OH
HO
Approved in 2001 for invasive aspergillosis



Glarea lozoyensis
O
NH
HO
O
Semi-synthetic, fungal
fermentation product
OH
O
Resistant to amphotericin B or triazole failure
Synergy: weakens cell wall and allows passage of
amphotericin B or fluconazole
2002 for esophageal candidiasis
Groll A. H.; Walsh T. J. Swiss Med. Wkly. 2002, 132, 303.
42
Echinocandin Summary

Different mechanism of action





No cross-resistance
Fungus must have cell wall
Minimal host toxicity
Minimal drug-drug interactions
Fungicidal
43
Future Targets
Moving into the cell
44
Promising Future Targets

Aspartate pathway


Fungi must synthesize Met, Ile, Thr
Siderophore biosynthesis

Iron importation mechanism
DeLaBarre B. et al. Nat. Struct. Biol. 2000, 7(3), 238.
Ferguson A. D. et al. Science 1998, 282, 2215.
45
Aspartate Pathway
O
O
O
O
H3N
O
Aspartate
ATP
AK
H3N
O
P O
O
O
O
O
Threonine
Isoleucine
O
NADH
ASD
Aspartyl-4Phosphate
H
O
H3N
O
Aspartate-4Semialdehyde
NADH
HSD
OH
O
H3N
O
Homoserine
AcCoA HSAT
O
AK = Aspartate Kinase
ASD = Aspartate Semialdehyde
Dehydrogenase
HSD = Homoserine Dehydrogenase
HSAT = Homoserine O-Acetyl
Transferase
O
O
H3N
O-Acetyl-
Homoserine
O
S
O
H3N
O
Bareich D. C. et al. Chem. Biol. 2003, 10, 967.
Methionine
46
Homoserine Dehydrogenase
Asp214
Thr176
O
O
O
H
H
H2N
Glu208
H
O
O
H
O
O
O
O
H
HN
H
H O
H
NH2
H
O
N
R
O
Lys223
DeLaBarre B. et al. Nat. Struct. Biol. 2000, 7(3), 238.
Asp219
NADH
47
Natural Product Inhibitor
O

Promising antifungal: 5-hydroxy-4oxonorvaline (HON)




OH
O
H3N
O
Isolated from Streptomyces over 40 yrs ago
Active against Cryptococcus and Candida
100% survival in rats, no toxicity
Ki = 2 mM; yet capable of arresting cell
growth (irreversible)
Jacques S. L. et al. Chem. Biol. 2003, 10, 989.
48
Mechanism of Inhibition
HON-NAD: biomolecular
mimic of 2 substrates
Lys223
Lys223
H3N
NH3
NH3
O
O
H3N
O
H3N
OH
O
H H
O
O
OH
N
R
O
NAD+
Jacques S. L. et al. Chem. Biol. 2003, 10, 989.
NH2
O
NH2
B
O
O
O
O
OH
NH2
N
R
N
R
49
Coupled Assay
O
O
O
H3N
O
O
AK
H3N
ATP ADP
O
O
P O
O
O
O
O
ASD
H
O
H3N
NADH NAD+
O
HSD
OH
O
H3N
O
NADH NAD+
O
HSAT
O
O
H3N
AcCoA CoASH
O
O
O
S
O2N
NO2
S
O
O
AK = Aspartate Kinase
ASD = Aspartate Semialdehyde
Dehydrogenase
HSD = Homoserine Dehydrogenase
HSAT = Homoserine O-Acetyl
Transferase
Bareich D. C. et al. Chem. Biol. 2003, 10, 967.
NO2 O
NO2 O
O
O
+
S
SSCoA
lmax = 412 nM
e = 13600 M-1 cm-1
50
Novel Inhibitors of AK
IC50 (mM)
Cl
OH
O
N
Cl
N
S
Cl
N
O
CF3
18 ± 3.7


O
N
H
N
2
3.1 ± 0.8

N
S
S

O
N
S
N
H
2a
3.6 ± 0.8
S
Reversible inhibitors
First non-amino acid
inhibitors of fungal AK
Leads to new
compound development
No effect on growth of
Candida species

N
N
Cl
N
H
S
N
Cl
N
H
1
O
N
S
N
H
Membrane transport or
efflux problems
N
N
N
2b
1.6 ± 0.7
N
Bareich D. C. et al. Chem. Biol. 2003, 10, 967.
51
Siderophore Function
O
O
NO
Fe O N
O O
N
H
N
HN
O
O
HN
NH
O

O

O
NH
H
N
O HO

ferricrocin

Ferric-hydroxamate
uptake (FhuA) protein
Ferguson A. D. et al. Science 1998, 282, 2215.
Winkelman G. Biometals 2002, 30(4), 691.
Fungi must
scavenge for iron
inside host
Siderophores bind
soluble iron with
high affinity
Actively transported
through cell wall
Couple antifungals
to iron-binding motif
52
sidA Required for Virulence



sidA encodes first committed step in
hydroxamate siderophore biosynthesis
DsidA: no growth in serum, no virulence in
animal model
Minimal host toxicity
HO
H O
H
O
NH2
NH2
+
NH2
N
R
O2
L-ornithine
HO
N5-oxygenase
Hissen, A. H. T. et al. Infect. Immun. 2005, 73(9), 5493.
Schrettl, M. et al. J. Exp. Med. 2004, 200, 1213.
O
O
NH2
N
H
OH
NH2
+
N
R
53
Conclusions



Invasive fungal infections remain a
complication of modern medicine
Urgent need exists for improved
antifungal agents
Extensive work is being done to validate
new targets and develop new drugs
54
Acknowledgments



Helen E. Blackwell
Blackwell group members
Practice talk attendees





Megan Jacobson
Katie Alfare
Jamie P. Ellis
Sarah Campbell
Jesse O’Neill
55
Allergic fungal sinusitis
Curvularae lunata
August 2002
1 week on amphotericin B
kidney failure
potassium levels
11 months on voriconazole
Racette A. J. et al. J. Am. Acad. Dermatol. 2005, 52(5), S81.
56