1 Recent Advances in Antifungal Drug Development Jennifer O’Neill February 2, 2006

1

Recent Advances in Antifungal Drug Development

Jennifer O’NeillFebruary 2, 2006

2

History Marketed Drug Classes

Polyenes Azoles Echinocandins

Future Targets Conclusions

Outline

3

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

4

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

5

Impact of Infections

21% 25%

35%

90%

Heart transplant patients die of invasive aspergillosis

Lung transplant patients die of invasive aspergillosis

Infection-related deaths in leukemia patients

HIV/AIDS patients will contract fungal infections

de Pauw, B. E.; Meunier F. Chemotherapy 1999, 45, 1.Image from DoctorFungus Corporation

6

Fungi Challenging to Target

Cellular similarities Complicates target identification

Diversity of structure Diversity of metabolic targets

Image from kvhs.nbed.nb.ca

Archaea

Eukaryotes

Bacteria

Fungi

AnimalsKIN

GD

OM

S Filamentous

Yeasts

7

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

8

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.

9

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

10

Polyenes

Binding ergosterol

O HO

OH

OHOH

OH

OHOHHO

H3C

CH3OHO

O

OH

OO

OHH2N

HO

11

Key Events in Polyene History

1940s 1950s 1960s 1990s

Sheehan, D. J. et al. Clin. Microbiol. Rev. 1999, 12(1), 40

1949First polyene identified:Nystatin

1956Amphotericin B activity reported

1960Amphotericin B approved

1990-92Lipid formulations of Amphotericin B introduced

1970s 1980s 2000s

12

Amphotericin B

Isolated from bacteria in 1956 Streptomyces noursei

The gold standard Most effective

antifungal for over three decades

Fungicidal Limited to fungi that

contain sterols

O

OH OH

HO

HO

OHHO

HO

OHH3C

CH3

OH

O

OHO

O

OHO

H2N

HO

13

OH

HO

HH

Mechanism of Action Amphotericin B

binds to ergosterol in cell membrane

Alters permeability of membrane

Ghannoum, M. A.;Rice L. B. Clin. Microbiol. Rev. 1999, 12(4), 501.Milhaud, J. et al. Biochim. Biophys. Acta 2002, 1558, 95.

ergosterol Amphotericin B

O HO

OH

OHOH

OH

OHOHHO

H3C

CH3 OHO

OH

O

OO

HO

NH2

OH

14

Mechanism of Action

Ghannoum, M. A.; Rice L. B. Clin. Microbiol. Rev. 1999, 12(4), 501.Milhaud, J. et al. Biochim. Biophys. Acta 2002, 1558, 95.

aggregatesOH

Aqueous pores cause leakage of vital cytoplasmic components

15

Drug of last resort – highly toxic

Resistance has been reported Fungi alter membrane composition

HOHOHH HH

Limitations of Amphotericin B

Ergosterol Cholesterol

vs.

FUNGAL MAMMALIAN

16

Azoles

Blocking ergosterol synthesis

NN

N X

X

R'

OR

17

Key Events in Azole History

1940s 1950s 1990s 2000s

1944First antifungal azole reported

1958First azole antifungal marketed: Ketoconazole

1990-92Fluconazole & Itraconazole introduced

1993-95Second generation triazoles reported

2005Posaconazole(Schering) approved

2002Voriconazole(Pfizer) approved


18

HOHO

Mechanism of Action

Inhibits cytochrome P450 14-demethylase

Fungistatic, not fungicidal

Lanosterolazole

s

NN

N X

X

R'

OR

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.

19

NN

NO

O O N N NN

N

OCl

Cl

NN

N F

F

N NN

OH

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

Fluconazole Itraconazole

20

Fluconazole

High safety profile – extensive use

Not active against Aspergillus spp.

Increasing reports of antifungal resistance

0

1

2

3

4

5

6

7

8

9

89 91 93 95 97

Year

Rate

of

Infe

ctio

n* C. albicans

non-albicans

*blood stream infections/10,000 central venous catheter days

NN

N F

F

N NN

OH

0

20

40

60

80

100

92 93 94 95 96 97 98 99

YearPro

port

ion (

%)

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.

21

NN

N

O O N N NN

N

OF

F

HONNN

F

FOH

N

NF

2nd Generation Triazoles Enhanced potency (10–500x) over 1st generation Broad-spectrum activity: yeasts, molds,

Aspergillus Excellent central nervous system penetration Greatly reduced toxicity

Voriconazole Posaconazole

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.

22

Derivatives of Fluconazole

N

NN

OHF

FR1

YX

R2

R3

Wanted to increase spectrum of activity to include Aspergillus spp.

Dickinson R. F. et al. Bioorg. Med. Chem. Lett. 1996, 6(16), 2031.

R1 = H, Me R2 = H, F, ClR3 = H, ClX =N, CHY = N, CH

OC2H5

O

FO

O

N

FNH

Cl

N

FN

N

FN

HN

H2NH

MeONa POCl3

reflux

H2, Pd/C

EtOH, 20 °C

Synthesis of fluoropyrimidine

23

NN

NO

O O N N NN

N

OCl

Cl

In vitro Activity of Azoles

MIC (g/mL)*

Flu Itr VorAspergillus 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

Cryptococus neoformans

9.6 0.39 0.39Itraconazole (Itr)

Fluconazole (Flu) Voriconazole (Vor)

NNN

F

FOH

N

NF

NN

N F

F

N NN

OH

*minimum inhibitory concentration

Dickinson R. F. et al. Bioorg. Med. Chem. Lett. 1996, 6(16), 2031.

24

Voriconazole

-CH3 gives a marked increase in activity

Pyrimidine ring expands therapeutic window

Side effects Multiple drug-drug interactions

NNN

F

FOH

N

NF

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.

25

Drug-Drug InteractionsRifampin EfavirenzRifabutin BarbituratesPhenytoin Terfenadine HIV Protease Inhibitors AstemizoleNNRTIs SirolimusCisapride PimozideQuinidine Ergot AlkaloidsCyclosporine MethadoneTacrolimus WarfarinOmeprazole BenzodiazepineVinca AlkaloidsHMG-CoA Reductase InhibitorsSulfonylurea Oral HypoglycemicsDihydropyridine Calcium Channel Blockers

Pfizer Inc. VFEND® Complete Product Information, March 2005.

26

Quantitative SAR Study

No 3-D structural data available in Candida Homology and pharmacophore modeling 5 structure classes: A–E

Di Santo R. et al. J. Med. Chem. 2005, 48, 5140

N

N

N

R

Cl

Cl N

N

MeO

RN

N

N

N

N

N R1

R

R1

N

N

N

R

R2

AB C

D E

27

O

N

H

Cl

Cl

CHOO

Cl

Cl

O

N

R

Cl

Cl

HO

N

R

Cl

Cl

O

Cl

Cl

C NTs

N

N

N

R

Cl

Cl

Synthesis of Class A

O

N

N

N

N

NaOH R-I, K2CO3

DMF

LiAlH4

EtOH NaHDMSO, Et2O

THF MeCN


28

In Vitro Anti-Candida Activity Tested in 12 Candida albicans strains


N

N

N

R

Cl

Cl N

N

MeO

RN

N

N

N

N

N R1

R

R1

N

N

N

R

R2

AB C

D E

MIC = 0.74–3.9 g/mL 3.5–340 g/mL 24 g/mL

2.5–26 g/mL 0.07–220 g/mLFluconazole0.24 g/mL

29

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

4UNA = unsubstituted Ar N EV = excluded volumesHY = hydrophobic RA = aromatic ring


30

Activity Prediction

N

N

N

R

Cl

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

Flu 0.069 0.59 8.6

N

N

Values expressed as MICcmpd/MICbifbifonazole

Class E

NN

N F

F

N NN

OH

fluconazole

Calc/

Expt


31

Azole Summary

2nd generation targets resistant strains

Broad spectrum activity Far less toxic than amphotericin B Multiple drug-drug interactions Fungistatic

32

Echinocandins

Targeting the fungal cell wall

OH

NH

HO

HNO

OH

NO

HN

OH

HO

HO

O

ONH

ON

OOH

HO

H3C

HO

NH

O

33

Key Events for Echinocandins

1940s 1950s 1960s 1990s 2000s

1988First echinocandin tested

2001Caspofungin(Merck) approved


34

Mechanism of Action

Image from DoctorFungus CorporationSawistowska-Schroder E. T. et al. FEBS Lett. 1984, 173(1), 134.

(1,3)glucan synthase

Phospholipid bilayerof cell membrane

Chitin

(1,6)-glucan(1,3)-glucan

Mannoproteins

Non-competitive inhibitors of (1,3)-glucan synthase

OHO

HO O P

OH

O

O P

OH

O

OO

HO OH

N

NH

O

OO

HOO O

OHO

HO

OHO

OO

HOHO

OHO

O O

OHOHOHOHOHOH

OHOH OH OH OH OH+

Cellwall

35

Echinocandins

Fungicidal Causes rapid lysis in growing cells

Candida & Pneumocystis carinii activity

Fewer drug-drug interactions Three in clinical development:

Caspofungin, micafungin, anidulafungin

OH

NH

HO

HNO

OH

NO

HN

OH

HO

HO

O

ONH

ON

OOH

HO

H3C

HO

NH

O

Letscher-Bru, V.; Herbrecht R. J. Antimicrob. Chemother. 2003, 51, 513.

36

SAR of Simplified AnalogsOH

NH

HO

HNO

OH

NO

HN

OH

HO

HO

O

ONH

ON

OOH

HO

H3C

HO

NH

NH

HNO

OH

NO

HN

O

ONH

ON

O

HO

NH

HO

O

R

O

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

R=

simplify

37

OH

NH

HO

HNO

OH

NO

HN

OH

HO

HO

O

ONH

ON

OOH

HO

H3C

HO

NH R

O

O(CH2)4CH3

O(CH2)7CH3

O(CH2)7CH3

NH

OR'

R'O

Sidechain SAR Study

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

R = -(CH2)n-CH3

n=11–21

R’ = -(CH2)n-CH3

n=5–13

R’ = -(CH2)n-CH3

n=6–15

(cilofungin)

(o, m, p)

38

OH

NH

O

HNO

OH

NO

HN

OH

HO

HO

O

ONH

ON

OOH

HO

HO

NH

O

H3N OC7H15

H

OH

NH

HO

HNO

OH

NO

HN

OH

HO

HO

O

ONH

ON

OOH

HO

HO

NH

O

H2N

O

H

H3N

Cationic Derivatives

Cilofungin withdrawn due to toxicity of solubilizing agent

Increase water solubility

Unique regio-, chemo-, and stereoselective synthesis from core 4 linear steps 83% yield

Pneumocandin B

Bouffard, F. A. et al. J. Med. Chem. 1994, 37, 222.Journet, M. et al. J. Org. Chem. 1999, 64, 2411.

39

OH

NH

HO

HNO

OH

NO

HN

OH

HO

HO

O

ONH

ON

OOH

HO

HO

NH

O

H2N

OH

NH

HO

HNO

OH

NO

HN

OH

HO

HO

O

ONH

ON

OOH

HO

HO

NH

O

H2N OC7H15

O O

Pneumocandin Semi-Synthesis

2. , TEA

1. enzymatic hydrolysis

Pneumocandin Bo isolated from Glarea lozoyensis Most efficient route began with acylation of amine

98%OC7H15

C6F5O2C

Journet, M. et al. J. Org. Chem. 1999, 64, 2411.

40

Dehydration and Etherification

OH

NH

HO

HNO

OH

NO

HN

OH

HO

HO

O

ONH

ON

OOH

HO

HO

NH R

O

H2N

OH

NH

O

HNO

OH

NO

HN

OH

HO

HO

O

ONH

ON

OOH

HO

HO

NH R

O

NCO

CbzHN

Direct reduction of amide gave mixture of products Protection of benzylic alcohol required

1. cyanuric chlorideDMF/H2O, -30 °C

OC7H15

R=

2. PhB(OH)2

CbzHNOH

3. CCl3CO2H

92%(99:1 /)

4. H2O


41

One Pot Hydrogenation Hydrogenation of nitrile Deprotection of Cbz-protected amine

OH

NH

O

HNO

OH

NO

HN

OH

HO

HO

O

ONH

ON

OOH

HO

HO

NH R

O

NC

OH

NH

O

HNO

OH

NO

HN

OH

HO

HO

O

ONH

ON

OOH

HO

HO

NH R

O

H2N

H2N

CbzHN

5 mol % Pd/Al2O3

10 mol % Rh/Al2O3

H2 (40 psi), 25 °C35 eq NH4OAc5% HOAc

92%

OC7H15

R=


42

Caspofungin

Semi-synthetic, fungal fermentation product

Glarea lozoyensis Approved in 2001 for invasive aspergillosis

Resistant to amphotericin B or triazole failure Synergy: weakens cell wall and allows passage

of amphotericin B or fluconazole 2002 for esophageal candidiasis

OH

NH

NH

HNO

OH

NO

HN

OH

HO

HO

O

ONH

ON

OOH

HO

HO

NH

O

H2N

H2N

Groll A. H.; Walsh T. J. Swiss Med. Wkly. 2002, 132, 303.

43

Echinocandin Summary

Different mechanism of action No cross-resistance

Fungus must have cell wall Minimal host toxicity Minimal drug-drug interactions Fungicidal

44

Future Targets

Moving into the cell

45

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.

46

O

O

O

H3N

O

O

O

O

H3N

O

P O

O

OO

H

O

H3N

O

O

OH

H3N

O

O

O

H3N

O

O

O

S

H3N

O

Aspartate Pathway

Methionine

Aspartate Aspartyl-4-Phosphate

Aspartate-4-Semialdehy

de

Homoserine

O-Acetyl-Homoserine

ATP NADH NADH

AcCoA

AK ASD HSD

HSAT

AK = Aspartate KinaseASD = Aspartate Semialdehyde

DehydrogenaseHSD = Homoserine DehydrogenaseHSAT = Homoserine O-Acetyl

Transferase

ThreonineIsoleucine

Bareich D. C. et al. Chem. Biol. 2003, 10, 967.

47

Homoserine Dehydrogenase

O

O

O

H

HN

Lys223

O

O

Asp219

N

O

NH2

H H

R

H2N

O

O

Glu208

Asp214O

OThr176 O

OHH

HH

H

H

NADH

DeLaBarre B. et al. Nat. Struct. Biol. 2000, 7(3), 238.

48

Natural Product Inhibitor

Promising antifungal: 5-hydroxy-4-oxonorvaline (HON) 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)

O

O

H3N

O

OH

Jacques S. L. et al. Chem. Biol. 2003, 10, 989.

49

Mechanism of Inhibition

Lys223

NH3

O

O

H3N O

N

NH2

O

R

H H

Lys223

NH3

O

O

H3N O

OH

N

NH2

O

R

B

O

O

H3N O

OH

N

NH2

O

R

OH

HON-NAD: biomolecular mimic of 2 substrates

NAD+

Jacques S. L. et al. Chem. Biol. 2003, 10, 989.

50

O

O

O

H3N

O

O

O

O

H3N

O

P O

O

OO

H

O

H3N

O

O

OH

H3N

O

O

O

H3N

O

O

SS

O2N

OO

NO2

OO

NO2

O

O NO2 O

O

SSCoAS

Coupled Assay

+

AK ASD HSD HSAT

ATP ADP NADH NAD+ NADH NAD+ AcCoA CoASH

max = 412 nM = 13600 M-1 cm-1

AK = Aspartate KinaseASD = Aspartate Semialdehyde

DehydrogenaseHSD = Homoserine DehydrogenaseHSAT = Homoserine O-Acetyl

Transferase


51

Novel Inhibitors of AK

Reversible inhibitors First non-amino acid

inhibitors of fungal AK Leads to new

compound development

No effect on growth of Candida species

Membrane transport or efflux problems

NNH

NH

O

CF3

Cl

OH

Cl

N

S S NH

O

N

Cl

ON N

N

S S NH

O

Cl

N N

S

N

S S NH

O

Cl

N N

NN

N

1 18 ± 3.7

2 3.1 ± 0.8

2a 3.6 ± 0.8

2b 1.6 ± 0.7

IC50 (M)


52

Siderophore Function 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

NH

HN

HN

HN

NH

HN

O

O

O

NON O

NOO

O

O

O

O

Fe

O

HO

Ferguson A. D. et al. Science 1998, 282, 2215.Winkelman G. Biometals 2002, 30(4), 691.

ferricrocin

Ferric-hydroxamate uptake (FhuA) protein

53

sidA Required for Virulence

sidA encodes first committed step in hydroxamate siderophore biosynthesis

sidA: no growth in serum, no virulence in animal model

Minimal host toxicity

HO

O

NH2

NH2

HO

O

NH2

NH

OHN

O

NH2

H H

R

N

O

NH2

R

+ +O2

L-ornithine N5-oxygenase

Hissen, A. H. T. et al. Infect. Immun. 2005, 73(9), 5493.Schrettl, M. et al. J. Exp. Med. 2004, 200, 1213.

54

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

55

Helen E. Blackwell Blackwell group members Practice talk attendees

Megan Jacobson Katie Alfare Jamie P. Ellis

Sarah Campbell Jesse O’Neill

Acknowledgments

56

Allergic fungal sinusitis

Racette A. J. et al. J. Am. Acad. Dermatol. 2005, 52(5), S81.

Curvularae lunataAugust 2002 1 week on amphotericin B

kidney failurepotassium levels

11 months on voriconazole

Documents

1 Recent Advances in Antifungal Drug Development Jennifer O’Neill February 2, 2006