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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
CO2 + H2O ↔ HCO3- + H+
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.)
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
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
.
α-CA       β-CA
(hCA II)   (Can2)




Blue=
Hydrophobic
Red = Hydrophilic
Yellow = metal



γ-CA       ζ-CA
(Cm)       (R3-CdCA)
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
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.
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
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
Sulfonamides/sulfamates
  used clinically (more than
  30 compounds)


Supuran, CT. Nature Rev
Drug Discov 2008, 7, 168-181
Supuran, CT. Nature Rev Drug Discov 2008, 7, 168-181
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
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)
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)
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)
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)
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 !)
hCA II – topiramate adduct (Casini et al., Bioorg. Med. Chem. Lett. 2003, 13
841-845)
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.
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.
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
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)
Principles of 3D Fitting of Molecules
Principles of 3D Fitting of Molecules
The hCA IX – acetazolamide adduct (Alterio et al., PNAS 2009)
hCA IX is a homodimeric protein
Crystal structure of hCA IX ( Alterio et al., Proc.
Natl. Acad. Sci. USA 2009, 106 , 16233-16238)
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.
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)
Collab. with
Univ of Maastricht




HT-29 bearing mice
injected with
fluore-
scent compounds

Dubois et al.,
Radiother. Oncol.
2009, 92, 423-428
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)
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).
Principles of 3D Fitting of Molecules
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
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
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.)
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.
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
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).
COOH
                                            HO -

                                    O   O          OH




hCA II – unsubstituted “coumarin”
adduct (TRANS-2-hydroxy-
cinnamic acid
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
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)
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
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
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.)
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
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).
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
Principles of 3D Fitting of Molecules
Superposition of the hCA II – acetazolamide complex with
the hCA II/IX mimic – sulfocoumarin complex (black)
POLYAMINES as CAIs: hCA II – spermine adduct
(Carta et al, J Med Chem 2010)
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.
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
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
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
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
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)
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
Principles of 3D Fitting of Molecules
Fluvastatin is 3
Atorvastatin is 4
Rosuvastatin is 5
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)
Active site details of Can2
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
Modeling of AAZ binding to C. albicans beta-CA (Nce103)
Principles of 3D Fitting of Molecules

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Principles of 3D Fitting of Molecules

  • 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
  • 2. CO2 + H2O ↔ HCO3- + H+
  • 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
  • 12. 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)
  • 27. hCA IX is a homodimeric protein
  • 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)
  • 52. POLYAMINES as CAIs: hCA II – spermine adduct (Carta et al, J Med Chem 2010)
  • 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
  • 61. Fluvastatin is 3 Atorvastatin is 4 Rosuvastatin is 5
  • 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
  • 65. Modeling of AAZ binding to C. albicans beta-CA (Nce103)