Lecture by Dr. Supuran in the context of the Course: "Tumour Hypoxia: From Biology to Therapy III".
For the complete e-Course see http://www.myhaikuclass.com/MaastroClinic/metoxia
1. PhD-course in Maastricht on the
Topic:Tumour Hypoxia: From Biology
to Therapy III
Principles for 3D-fitting of molecules to an
enzyme - measuring side-effects of drugs:
carbonic anhydrases as an example
Claudiu T. Supuran
University of Florence, Italy
claudiu.supuran@unifi.it
3. 5 Carbonic anhydrase (CA) gene families
α-CAs (Bacteria, algae, cytoplasm of green plants, protosoa
(e.g. Plasmodium), animals – including vertebrates)
β-CAs (Bacteria, algae, chloroplasts of mon-/dicotyledons)
γ-CAs (Archaea, Bacteria)
δ- CAs – marine diatoms and algae (e.g., Thalassiosira
weissflogii TWCA1 and related organisms)
ζ- CAs –Cd or Zn enzymes from marine diatoms
These gene families are evolutionary unrelated.
(Supuran, CT. Bioorg Med Chem Lett 2010, 20, 3467-3474.)
4. α-CAs in higher vertebrates including Homo sapiens
___________________________________________________________________________
Isozyme Catalytic activity Affinity Sub-cellular localization
(CO2 hydration) for sulfonamides
__________________________________________________________________
CA I medium medium cytosol
CA II high very high cytosol
CA III very low very low cytosol
CA IV high high plasma membrane
CA VA moderate high mitochondria
CA VB high high mitochondria
CA VI medium high secreted (saliva/milk)
CA VII high very high cytosol
CA VIII acatalytic - cytosol
CA IX high high transmembrane
CA X acatalytic - cytosol
CA XI acatalytic - cytosol
CA XII medium very high transmembrane
hCA XIII low high cytosol
hCA XIV low high transmembrane
mCA XV high high plasma membrane
________________________________________________________________________________________
h = human, m = murine isoforms
5. hCA II active site, with the Zn(II) ion (pink sphere), its three histidine ligands
(His 94, His 96 and His 119, in green), the proton shuttle residue His 64 as well
as the histidine cluster extending from the rim of the active site to the surface
of the protein, comprising residue 3, 4, 10, 15 and 17, in orange)
Supuran, CT. Nature Rev Drug Discov 2008, 7, 168-181
.
6. α-CA β-CA
(hCA II) (Can2)
Blue=
Hydrophobic
Red = Hydrophilic
Yellow = metal
γ-CA ζ-CA
(Cm) (R3-CdCA)
7. Catalytic mechanism of α-CAs
Hydrophobic pocket
Val 121
Val 143
- Leu 198
OH O
-
+ CO2 OH O
2+
Zn 2+
His 119 Zn
His 94 His 119
His 96 His 94
His 96
A
B
- BH+ B
OH2 O
H
Zn
2+ O
+ H2O -
His 119 O
His 94 Zn
2+
His 96 - HCO -
3 His 119
His 94
His 96
D
C
8. CA inhibition mechanism (by sulfonamides (a) and anions (b),
cf. Supuran, CT. Nature Rev Drug Discov 2008, 7, 168-181;
Supuran, CT. Bioorg Med Chem Lett 2010, 20, 3467-3474.
9. Sulfonamide CAIs in clinical use :
Classical use (since 1954):
1. Diuretics
2. Antiglaucoma systemic drugs
“Modern” use/applications:
1. Topical antiglaucoma drugs
2. Anticonvulsants/antiepileptics
3. Antiobesity agents
4. Antitumor therapies/diagnostic tools
5. Anti-infectives
Different isozymes/enzyme classes are targeted by
such drugs
10. Inhibitors designed – novel classes of CAIs
Hystoric overview on CAI drug discovery
1940 1954 1987 1992 2002 2008-9 2010 2011
Sulfamates Sulfamates
DITHIO-
(topiramate) as CAIs COUMARINS
Acetazolamide CARBAMATES
reported not to(Maren)
starts to be used
be CAIs
clinically
(Maryanoff) POLYAMINES
Sulfamides
Sulfonamides discovered (Winum et al)
as CAIs
11. Sulfonamides/sulfamates
used clinically (more than
30 compounds)
Supuran, CT. Nature Rev
Drug Discov 2008, 7, 168-181
13. Sulfonamide diuretics revisited (Temperini et al., Bioorg. Med. Chem. 2009)
Cl
Cl O
H Me W142 H 2.70 O N
N H H
O SO2NH2
N H Asn67 NH
OH N SO2NH2 2.83 O NH2
2.65 O 3.76
O H N O
W146
N H His64 (in conformati
3.1-3.4 N
Chlorthalidone O H H
O H
Indapamide NH 3.16 Phe131
H 2.54 H
Cl O W161
H H
Cl N Cl N Cl O S Cl
Thr200 O S Cl O
O O -
HN - HN 3.19 NH 3.06
HN 2.92 NH 2.89
S SO2NH2 HOOC SO2NH2 2+
2.15 Zn
2+
O O 2.02 Zn
O 2.77 His119 O 2.86 His119
Trichloromethiazide Thr199 Thr199
Furosemide H H
His96
His94 His96 His94
W2108 B
O A
H
H 3.18 W2201
His64
2.70
His64 O
Cl H
Cl H W2043 H 2.66 O
O NH 2.66 O
NH 3.15 H 2.86
H -
N N HN
N
O NH 3.02 O
S O
O O H 3.19
3.04
O H 2.85 Thr200 H
Thr200 NH2
W2048 O H
Gln92 O S Cl
2.92 O S Cl O
O -
- HN 2.97 NH 2.96
HN NH 3.04
2+
2.00 Zn
2+
2.04 Zn
O 2.95 His11 Thr199 O 2.94 His119
Thr199 H H
His96
His94 His96 His94
C D
14. CA II inhibition
Ki(CTD) = 138 nM
Ki(IND) = 2520
nM
Ki (TCM) = 91 nM
Ki(FUR) = 65 nM
Temperini et al.
Bioorg. Med. Chem.
2009, 17, 1214-21.
But, other isoforms are
inhibited better
CTD: 9 nM (CA VB)
IND: 10 nM )CA XII)
TCM: 8 nM (CA VII)
hCA II – chlorthalidone adduct (yellow): indapamide adduct (wheat)
Trichloromethiazide adduct (sky); furosemide adduct (magenta)
15. Antiglaucoma CAIs : a novel approach - NO-donating sulfonamides
Derivatization of
carboxy/amino-
sulfonamides with NO-donating
moieties
Supuran et al, WO2008/071421
Mincione et al., BMCL 2011
Nanomolar CA II, IV and XII
inhibitors (10-50 nM)
(the isoforms targeted by anti-
glaucoma CAIs)
16. X-ray crystal structure of the hCA II – C1 adduct
O
O
O N N
S N S SO2NH2
O H
O
O2N
C1 Ki = 35 nM (CA II); 47 nM
(CA IV); 13 nM (CA XII)
17. 0
-2
1
-4
2
-6
∆ IOP (mm Hg)
3
-8
-10
-12
4
-14
-16
-18
0 1 2 3
Time (hours)
IOP lowering in glaucomatous rabbits
Curve: 1: dorzolamide 2 %; 2 – brinzolamide 2%; 3- NO-donating sulfonamide
C1 at 1 %; 4 - NO-donating sulfonamide at 2 %.
(initial IOP in the range of 38-43 mm Hg)
18. Anticonvulsant CAIs (De Simone et al., Chem. Biol. Drug. Des. 2009, 74, 317-21)
SO2NH2
N N SO2NH2
O S
O O
1: zonisamide 2: sulthiame
O
NH2
O S
O O
O
O
O
O
3: topiramate
Anticonvulsant sulfonamides/sulfamate
Low nanomolar inhiibtion of CA II, VII,
XII and XIV (among others)
CAs present in the brain (many !)
19. hCA II – topiramate adduct (Casini et al., Bioorg. Med. Chem. Lett. 2003, 13
841-845)
20. hCA II – zonisamide adduct
ZNS is a potent CAI: Ki = 35 nM (hCA II) and 20 nM (hCA V)
De Simone et al., Bioorg. Med. Chem. Lett. 2005, 15, 2315-20.
21. CA VII adduct with acetazolamide (Di Fiore et al, BMCL 2010)
BRAIN-associated isoform. Is it the target of anticonvulsants?
2 Cys residues from CA VII are glutathionylated (Monti et al, BMCL 2012)
Involved in oxidative stress.
22. Inhibition of mitochondrial CAs for obtaining antiobesity agents
Side effects of topiramate
or zonisamide treated
patients: loss of weight
CA VA/B inhibition
leads to fatty acid bio-
synthesis inhibition
TPM and ZNS used for
obesity
FDA approved TPM
(Qnexa) in Feb 2012
Supuran, CT. Nature Rev Drug Discov 2008, 7, 168-181
23. CA IX/XII as targets in the antitumor drug design
-CA IX: catalytically efficient isozyme (kcat = 5.5 .105 s-1); CA XII
shows lower activity (approx 10 times)
-tendency to form oligomers (dimers)
-tumor-associated, transmembrane isoforms
-highly overexpressed in hypoxic tumors (HIF-1alpha cascade activation)
-bad prognostic tumor markers (non responsiveness to chemo and radio-
therapy)
26. The hCA IX – acetazolamide adduct (Alterio et al., PNAS 2009)
28. Crystal structure of hCA IX ( Alterio et al., Proc.
Natl. Acad. Sci. USA 2009, 106 , 16233-16238)
29. Design of fluorescent sulfonamides as probes of tumor-associated CAs
CSCl2 HCl H2N A SO2NH2
H2O
NH2
N A SO2NH2
COOH + C
S S
HO O O
DMA, NEt 3 HN N A SO2NH2
H
COOH
DMF, NEt 3
S
C HO O O
N
+ H2 N B SO2NH2
COOH
HO O O
Cecchi et al., J Med Chem 2005, 48, 4834 - 4841
Low nanomolar inhibitors of CA IX, XII and II.
30. Sulfonamide binding to hypoxic MDCK-CA IX cells and its effect on pHe
a
S
HN N
H
SO2NH2
COOH
HO O O
5
b
Normoxia Hypoxia Normoxia Hypoxia
hCA II KI= 44 nm
hCA IX KI= 26 nm
mock CA IX
(a) The sulfonamides (0.1 mM) were added to MDCK-CA IX cells before their
transfer to hypoxia and pHe was measured 48 h later. (b) Fluorescence analysis
of the transfected MDCK cells plated on the glass coverslips. The cells were
treated with the FITC-labelled compound 5 throughout the 48 h incubation in
normoxia and hypoxia, respectively (Svastova et al FEBS Lett. 2004, 577, 439-45)
31. Collab. with
Univ of Maastricht
HT-29 bearing mice
injected with
fluore-
scent compounds
Dubois et al.,
Radiother. Oncol.
2009, 92, 423-428
32. Gold nanoparticles coated with sulfonamide CAIs ( Stitti et al J. Am. Chem. Soc.,
2008, 130, 16130-1 )
SO2NH2
S OH
S SO2NH2
H2N HN
O
1 2
O
EDCI
DMAP
S S GNP-1
S H
N HAuCl4, 4H2O Au
S
O NaBH4, DMSO
3 SO2NH2
Compound Inhib const (nM)
hCA I hCA II hCA IX
Novel GNPs are
AZA 250±12 12±1 25±1
being presently
prepared and assayed as
3 214±9 230±10 41±2
CA IX/XII inhibitors
(Carta et al., in prep.)
GNP-1 581±18 (128) 451±21 (116) 32±2 (2.4)
33. Anticancer/antimetastatic action of the CAIs
Relevant progress achieved ultimately based on:
1. Svastova et al 2004
2. Ahlskog et al 2009 (Dario Neri’s group)
3. Dubois et al 2009, 2010
CAIs, of the sulfonamide, sulfamate or coumarin type, show a relevant
inhibition of the growth of the primary tumor and metastases in many
animal models of cancer (various solid tumors).
35. Specific Inhibition of CAIX-positive 4T1 Tumor growth by a CAIX
small molecule Inhibitor (Lou et al, Cancer Res. 2011, 71, 3364-76 )
4T1 67NR
CAIX CAIX
β-actin β-actin
75 mg/kg 75 mg/kg
Tumor volume (mm )
Tumor volume (mm )
150 mg/kg 150 mg/kg
Vehicle Vehicle
CAI 17
Untreated Untreated
*
*P<0.02
** **P<0.01
Rx start
Rx start Rx end Rx end
3
3
7 21
Days post tumor inoculation Days post tumor inoculation
Mouse weight (g)
Mouse weight (g)
75 mg/kg 75 mg/kg
150 mg/kg 150 mg/kg
Vehicle Vehicle
Untreated Untreated
Days of Rx Days of Rx
36. CAI17 inhibits the formation of lung metastases
by 4T1 mammary tumor cells
Lavoro no. 630
della lista
pubblicazioni
12 days post-iv injection of 4T1 cells
Untreated Vehicle
5 doses ip
25 mg/kg 75 mg/kg
37. MST-119 inhibits the formation of metastases by 4T1
mammary tumor cells
Day 7 post injection of tumor cells (5x10 5 cells/animal)
NO2
H2NO2S Pacchiano et al., J. Med. Chem. 2011, 54,
O
MST-119 1896-902.
N N
H H
Vehicle n=4 per group
*P<0.05
Total flux (X10 photons/sec)
Total flux (photons/sec)
15 mg/kg
6
*
*
45 mg/kg
3 doses (eod x 3 i.p.)
38. Coumarins as CAIs: HTS in collab with Griffith
Univ (Brisbane, Australia)
An australian plant as source, Leionema ellipticum Paul G. Wilson
(Rutaceae) allowed the isolation of a first such derivative (1)
OH COOH
O O O O O O O O O O O
2 3
1 4
Ki = 56 – 99 nM (for 1-4) against hCA II
Ki = 78 nM – 6 uM (for 1-4) against hCA I
Ki = 54.5 – 767 nm (for 1-4) against hCA IX
Ki = 49 – 167 nM (for 1-4) against hCA XII
Maresca et al., JACS, 2009, 131, 3057-62.
39. OH OH
HO -
O O O COOH
O OH
Binding of the CIS-2-hydroxy-cinnamic
acid (in gold) hydrolysis product of the
coumarin NP within hCA II active site
40. Superposition of hCA II – 5 (coumarin hydrolysis product) – gold – with hCA II –
phenol adduct (sky) and hCA II – sulfonamide adduct (possessing a TEMPO tail)
(magenta).
41. COOH
HO -
O O OH
hCA II – unsubstituted “coumarin”
adduct (TRANS-2-hydroxy-
cinnamic acid
42. Coumarins: the most isoform-selective CAIs reported to date
OH OH
-
HO
-
COO
O O O O OH
A
A-Z
-
NaOH COO
-
COO
O - Na
+
O OH OH
B B-Z B-E
Maresca et al. JACS 2009,
J Med Chem 2010
43. The COUMARINS and their derivatives
R
O O S O
Lactones
(6-membered) Thiocoumarin
O S
2-Thioxocoumarin
O S
Thioactones
(6-membered)
R
R O O S S
COUMARIN Dithiocoumarin
O NOH
Lactone oximes
O
O O N OH
Lactones
(5-membered) R Coumarin-oximes
S O
10 O
O
different
"Sulfocoumarin"
new classes of CAIs
Chemical diversity alreday generated using coumarins as lead (all PRODRUG CAIs)
44. Glycosyl-substituted coumarins are low nM CA IX/XII inhibitors
Do not inhibit significantly CA I and II (Winum et al., J Med Chem 2011,
54, 8271-7)
OAc OAc OAc
OAc OAc OAc
O morpholine O Cl3CCN, DBU O
AcO AcO AcO
AcO OAc AcO OH AcO O
CH2Cl2 CH2Cl2 CCl3
1 2 3 HN
BF3.OMe2 HO O O
CH2Cl2
4
OH OAc
OAc
HO O O
HO AcO
HO AcO
NaOMe
O O O MeOH O O O
6 5
in collaboration with Winum and Dedhar groups
45. Glycosyl-substituted couymarins have high antitumor and antimetastatic activity
(Winum et al., J Med Chem 2011; Lou et al, 2011)
Treatment with a CA IX-selective glycosyl coumarin 6 attenuates the growth of
mouse breast tumors. 4T1 murine mammary tumor cells were implanted orthotopically
into BALB/c mice and tumors were grown for 14 days. Animals then received the
indicated doses of glycosyl coumarin 6 daily by i.p. injection
46. 6 inhibits the formation of metastases by 4T1 mammary
tumor cells (Lou et al 2011)
Day 7 post injection of tumor cells (2x105 cells/animal)
Vehicle
n=8 per group
*P<0.004
Total flux (X10 photons/sec)
**P<0.001
Total flux (photons/sec)
15 mg/kg
6
**
30 mg/kg *
5 doses (qd x 5 i.p.)
47. Isoform selective CAIs based on the coumarins (Maresca et al., J Med Chem 2010)
COOH OH
COOH COOH
MeO O O O O O O
11 OMe
15
16
Ki = 48 nM (CA XIII)
Ki = 99 nM (CA II) Ki-s > 5 uM all other CAs Ki = 1 uM (CA XIV)
Ki-s > 4 uM all other CAs Ki-s > 6 uM (all other CAs)
OH
COOH
COOR
R
S O
O O O O
17
19: R = Et
22: R = NH2
Ki = 47 nM (hCA IX)
kI = 42 nM (CA XIII) Ki = 48 nM (CA IV)
Ki = 45 nM (CA VII) Ki = 40 nM (CA XIII)
Ki-s > 4 uM (other CAs)
Ki = 47 nM (CA IX) Kis > 4 uM all other CAs
Ki = 46 nM (CA XV)
Kis > 3 uM other CAs
N
N N
N
O O
23
Ki = 48 nM (CA IX)
Ki-s > 3 uM all other CAs
48. hCA II – 3 adduct (gold), hCA II – 4b adduct (violet) ; 3 = cis acid, 4 = trans acid,
hCA II – phenol adduct (sky blue) and hCA II – sulfonamide adduct (magenta).
49. Sulfocoumarins as CAIs
O H O H
-
H O
-
CO O
O O O O O H
1
3Z
-
N aO H CO O
-
CO O
O -N a
+
O O H O H
2
4Z 4E
R R
H 2O R S O 3H
S O
S O 3H
O O H
O O H
51. Superposition of the hCA II – acetazolamide complex with
the hCA II/IX mimic – sulfocoumarin complex (black)
53. Spermine Ki-s (uM)
hCA I: 230
hCA II : 84
hCA III: 167
hCA IV: 10 nM
hCA VA: 0.84
hCA VB: 0.83
hCA VI: 0.99
hCA VII: 0.71
hCA IX: 13.3
hCA XII: 27.6
hCA XIII: 22.5
hCA XIV: 0.86
mCA XV: 74
Superimposition of the spermine (yellow), phenol (magenta)
and trans-2-hydroxycinnamic acid (violet) adducts with hCA II.
The Zn(II) ion is the violet sphere.
54. Novel Chemotypes: Dithiocarbamates (DTCs) – a new class of CAIs
Trithiocarbonate (CS3)2- was
shown recently to be a CAI
(Innocenti et al. BMCL19 (2009)
1855-1857
TTC is thus a NEW ZBG
(zinc-binding group)
TTC is however a weak or very weak CAI, Ki-s in the micro-millimolar range
(depending on the isoform)
CAN we design more effective CAIs considering TTC as lead ? YES
55. DTCs contain the ZBG foun in TTC:
Amine + CS2 + base →R1R2N-CSS-M+
1-27 1a-27a
We tested the commercially available dimethyl- and diethyl-DTC and found them
to be micromolar CAIs (more effective than TTC) and then synthesized more com-
plicated molecules.
N S
N S
S
S
Dimethyl-DTC Diethyl-DTC
PRIMARY and SECONDARY amines included in the study
56. CAI, II, IX and XII inhibition data with newly prepared primary DTCs
(Carta et al., J Med Chem 2012)
R1R2N-CSS-M+
1a-10a
Cmpnd R1 R2 KI (nM)
hCA I hCA II hCA IX hCA XII M
1a H Ph 4.8 4.5 4.2 4.3 K
2a H O[(CH2CH2)]2N 4.8 3.6 29.1 9.2 K
3a H MeN[(CH2CH2)]2N 33.5 33.0 22.1 17.5 K
4a H 2-butyl 21.1 29.4 4.6 31.7 K
5a H O[(CH2CH2)]2N(CH2)2 31.8 36.3 4.5 4.2 K
6a* H N[(CH2CH2)N]3 31.9 13.5 27.4 9.3 K
7a H PhCH2 4.1 0.7 19.2 11.5 Na
8a H 4-PyridylCH2 3.5 16.6 26.0 24.1 Et3NH
9a H [(CH2)5N]CH2CH2 4.5 20.3 3.6 20.5 K
10a H 2-thiazolyl 3.9 4.6 12.6 22.0 Et3NH
57. CA I, II, IX and XII inhibition data with newly prepared secondary DTCs
R1R2N-CSS-M+ Carta et al., J Med Chem
13a-27a 2012
Cmpnd R1 R2 Ki (nM)
hCA I hCA II hCA IX hCA XII M
13a Me Me 699 6910 714 798 Na
14a# Et Et 790 3100 1413 1105 Na
15a (CH2)5 0.96 27.5 70.4 46.1 Na
16a iso-Bu iso-Bu 0.97 0.95 4.5 0.99 Na
17a n-Pr n-Pr 1838 55.5 53.8 7.0 Na
18a n-Bu n-Bu 43.1 50.9 50.3 5.8 Na
19a n-Hex n-Hex 48.0 51.3 27.4 16.1 Na
20a Et n-Bu 157 27.8 25.9 7.5 Na
21a HOCH2CH2 HOCH2CH29.2 4.0 4.3 4.2 Na
22a Me Ph 39.6 21.5 28.2 7.7 Na
23a Me PhCH2 69.9 25.4 53.0 3.0 Na
24a O[(CH2CH2)]2 0.88 0.95 6.2 3.4 Na
25a NaS(S=C)N[(CH2CH2)]2 12.6 0.92 37.5 0.78 Na
26a (NC)(Ph)C(CH2CH2)2 48.4 40.8 757 169 Na
27a** (S)-[CH2CH2CH2CH(COONa)]2.5 17.3 4.1 4.0 Na
58. X-ray crystallography of hCA II adducts ith 3 new DTCs
Carta et al., Chem Comm 2012, 48,
1868-70.
B
A
A: PhCH2-Me-DTC 23a, Ki (CA2) = 25 nM
C B: morpholine-DTC 24a
C: 4-Ph-4-cyano-cycloheylamine-DTC 26a
24a and 26a are subnanomolar CA2 inhibitors
(X-ray done by McKenna’s group)
59. Superposition of the three hCA II – DTCs X-ray structures showing a very
variable orientation/conformation of the bound inhibitors within the active site
In collaboration with Rob McKenna’s group
62. Antifungals
CO2 sensing system is present in pathogenic fungi (Muhlschlegel et al, 2005)
For example, the regulation of C. neoformans capsule biosynthesis
(Supuran, Nat. Rev. Drug Discov. 2008, 7, 168)
64. Antifungals ? C. neoformans, C. albicans, C. glabrata, S. cerevisiae CAs
C. neoformans CA (Can2 ) X-ray crystal structure
Steegborn et al., J Mol Struct 2009