The η-class carbonic anhydrases as drug targets for antimalarial agents.

Review

The h-class carbonic anhydrases as drug targets for antimalarial agents 1.

Introduction

2.

Amino acid sequence analysis of P. falciparum CA

3.

Phylogenetic analysis of PfCA and related enzymes from

Expert Opin. Ther. Targets Downloaded from informahealthcare.com by Gazi Univ. on 02/05/15 For personal use only.

Plasmodia 4.

Kinetic properties of PfCA

5.

PfCA inhibition studies

6.

Expert opinion

Claudiu T Supuran & Clemente Capasso† †

Istituto di Bioscienze e Biorisorse (IBBR)- CNR, Napoli, Italy

Introduction: The h-class of carbonic anhydrases (CAs, EC 4.2.1.1) was recently discovered as the sixth genetic family of this metalloenzyme superfamily, and seems to be present only in various Plasmodium species, the malariaprovoking pathogens. The present review through detailed biochemical, kinetic and phylogenetic studies afford a clear view regarding the differences between h- and the other CA families. Areas covered: In this review, the authors underlined as the h-CAs, like a-, gand d-class enzymes, have the Zn(II) ion coordinated by three histidine residues and a water molecule. They seem to be more closely related to the aCAs, but there are notable differences between them, such as the lack of the proton shuttle residue (His64) and gatekeeper residues, Glu106 and Thr199 in the h-CAs, which are conserved in all a-CAs. Expert opinion: Plasmodium falciparum h-CA showed a moderate but significant activity for the CO2 hydration reaction, with a kcat of 1.4 105s-1 and a kcat/Km of 5.4 106 M-1 s-1. Several inhibition studies with anions and sulfonamides/sulfamates, allowed the identification of interesting lead compounds. The discovery of h-CA-specific inhibitors may lead to novel such agents with a new mechanism of action. Keywords: h-class enzyme, anion inhibitor, antimalarials, carbonic anhydrase, drug target, sulfonamide Expert Opin. Ther. Targets [Early Online]

1.

Introduction

Malaria is a mosquito-borne infectious disease of humans and other animals caused by parasitic protozoans belonging to Plasmodium spp. Plasmodia that can infect humans are represented by five species: Plasmodium falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi [1-7]. Plasmodia have the ability to move from intestinal cells towards liver cells through the portal circulation. When colonizing the liver tissue, plasmodia complete their vital cycle, and spread into the systemic circulation of their host [8-10]. Parasite life cycle is reactivated when a hematophagous arthropod insect (for example the mosquito), by stinging its relevant host, transfers the infectious plasmodia to another individual, who becomes infected in this way [11,12]. The primary treatment of malaria consists in a rapid and complete elimination of the Plasmodium parasite from the patient’s blood in order to prevent progression of uncomplicated malaria to severe disease or death, and to chronic infection that leads to malaria-related anemia and other complications. For this scope, several classes of antimalarial drugs are used, most of which have been developed decades ago, and relevant drug resistance problems emerged against most of them [13-15]. The clinically used antimalarials include among others the natural product quinine, the widely used chloroquine and its analogs such as amodiaquine and primaquine, proguanil, the sulfa drug sulfadoxine and pyrimethamine, as well as another natural

10.1517/14728222.2014.991312 © 2014 Informa UK, Ltd. ISSN 1472-8222, e-ISSN 1744-7631 All rights reserved: reproduction in whole or in part not permitted

1

C. T. Supuran & C. Capasso

Article highlights. . . . . .

A new class of CA was recently discovered in Plasmodium falciparum and other protozoa. The new CA class was named with the greek letter h. h-CAs seem to be more closely related to a-CAs, but there are notable differences between them. Plasmodium falciparum h-CA showed a moderate but significant activity for the CO2 hydration reaction. Inhibition studies with anions and sulfonamides/ sulfamates allowed the identification of interesting compounds.


This box summarizes key points contained in the article.

product, artemisin (Chart 1) [4,5]. These drugs were developed in the ‘50s and ‘60s and no new antimalarial agents were thereafter discovered for decades. The lack of effective vector control or vaccine, the limitation and high toxicity of the antimalarial drugs mentioned above, as well as the spread of drug-resistance malaria plasmodia has prompted scientists to search for novel anti-malarial drugs which should be more effective, less toxic and with a different mechanism of action compared to the clinically used agents [11,12,16-18]. In this context, the knowledge of the Plasmodium metabolic pathway is of pivotal importance for chemotherapy. For example, it is known that the parasite utilizes purine and pyrimidines for DNA/RNA synthesis during its exponential growth and replication [17,19,20], and that plasmodia synthesize pyrimidines de novo from HCO3-, ATP, glutamine, aspartate and 5-phosphoribosyl-1-triphosphate. HCO3- is the substrate of the first enzyme involved in the Plasmodia pyrimidine pathway, being generated from CO2 through the action of the enzyme carbonic anhydrase (CA, EC 4.2.1.1) [17,19,20]. CAs are metal-containing enzymes that catalyze the reversible conversion of carbon dioxide to bicarbonate and protons (CO2 + H2O HCO3-- + H+) [21,22]. CAs are present in all life kingdoms, with six genetically distinct families reported so far: a-, b-, g-, d-, z- and h-classes [6,7]. Most of them are zinc containing enzymes, but Fe(II) is present within the active site of the g-CAs, (described in Bacteria, Archaea and plants), whereas Cd(II) or Zn(II) ions are equally effective for promoting catalysis in the z-CAs (present in diatoms) [6,23-36]. The metal ion from the enzyme active site is coordinated by three His residues (in the a-, g- d- and hclass enzymes) or by one His, and two Cys residues (in the b- and z-CAs), with the fourth ligand being a water molecule/hydroxide ion acting as nucleophile in the catalytic cycle of the enzyme [6,7,25,37-40]. Many classes of CA inhibitors (CAIs) are known, including various anions, azoles, phenols, hydroxyurea and structurally similar small molecules, carboxylates/hydroxamates, organic phosphates and phosphonates, and various sulfonamide derivatives (R-SO2-NH2), which represent the main 2

class of clinically used CAIs [6,41-48]. Many of the mammalian enzymes are targets for antiglaucoma, antiepileptic, or anticancer drugs as well as for some diuretics [6,18,36,41-50]. A rather large number of primary sulfonamides (and one sulfamate) have been in clinical use for a long time, mainly as antiglaucoma, antiepileptic, and diuretic agents. Such agents include acetazolamide, methazolamide, dichlorophenamide, dorzolamide, brinzolamide, topiramate, and so on see later in the text [41-50]. A truncated form of the P. falciparum CA gene was cloned, expressed and purified in 2004 by Krungkrai’s group, who showed that it is an active enzyme, possessing esterase activity with 4-nitrophenylacetate as a substrate, but its CO2 hydrase activity was not studied [19]. This enzyme was also inhibited by the known sulfonamide CAI acetazolamide. These authors thus concluded that the enzyme belongs to the a-CA class [19]. Subsequent studies from Krungkrai’s and other groups showed that different Plasmodium spp. encode CAs, all considered to belong to the a-class, and that primary sulfonamides inhibited in vitro and in vivo the growth of the parasite [11,12,17]. However, recently it has been shown that the enzymes mentioned above belong in fact to a new CA genetic family, the h-CA class [7]. In the present paper we detail the findings which led to the discovery of this new CA genetic family and its implications as drug target for the design of antimalarials with a novel mechanism of action. We analyzed the amino acid sequences belonging to different Plasmodium species and compared them with those of CAs from other organisms. Detailed phylogenetic analyses as well as biochemical measurements will be discussed, which prove that the h-CA class is distinct from the other CA classes investigated so far. Such data prompt us to propose that h-CAs are druggable targets. By analyzing literature data on the affinity of various inhibitors for such enzymes, it appeared obvious that there are significant differences between the mammalian and protozoan CAs, which might be exploited for the development of h-CA-selective inhibitors and presumably new antimalarials.

Amino acid sequence analysis of P. falciparum CA

2.

Genes encoding for CAs in protozoan parasites have been identified in Plasmodium, Theileria, Trypanosoma, Leishmania and Entamoeba. One of the causative agents of human malaria, P. falciparum, was one of the first to be investigated in detail [7,19]. The open reading frame of the malarial enzyme (P. falciparum CA, accession number AAN35994.2, PlasmoDB: PF3D7_1140000) encodes a 600 amino acid polypeptide chain (Figure 1). A truncated form of the P. falciparum CA gene (Figure 1, gray color) was cloned, expressed and purified recently [7]. It has been demonstrated that this is a catalytically active enzyme for the physiological reaction catalyzed by the CAs, that is, CO2 hydration to bicarbonate

Expert Opin. Ther. Targets (2014) 19(4)

The h-class carbonic anhydrases as drug targets for antimalarial agents

N

HN

N

Cl


Quinine

Chloroquine

Amodiaquine

Primaquine

O

O H N

H N NH

H N NH

Cl

O

O S

N H

N N

H2N

Sulfadoxine

Proguanil

Pyrimethamine

Artemisin

Chart 1. Clinically used antimalarial drugs.

and protons, and that this catalytic activity is inhibited by the sulfonamide inhibitor acetazolamide [7]. The results of BLASTp analysis [51,52], curried out using the P. falciparum CA as query amino acid sequence, showed that the first top ranking library sequences exhibit: i) high amino acid sequences identity with CA belonging to the other species of plasmodium; ii) 36% of identity when compared with Theileria species; and iii) an identity < 30% when the malarial enzyme was compared with the other protozoan enzymes. Furthermore, Plasmodium CA displayed a very low identity (ranging from 25 to 30%) with the human a-CA isoenzymes. The alignment of the amino acid sequences of the truncated form of the P. falciparum CA gene (PfCA) with the human a-CA (hCA I) is shown in Figure 2. PfCA sequence can be aligned to the zinc ion ligands of hCA I

only if gaps are added to the amino acid sequence regions in which the zinc-coordinating histidines are located (Figure 2). Using the hCA I numbering system, six gaps must be placed in the PfCA sequence (after residue 96), and five gaps in the hCA I sequence (after Leu118). On this basis, Plasmodia CAs were assigned to the a-CAs by Krungkrai’s group [19]. Our earlier publication [7] however demonstrated that Plasmodia CAs were erroneously assigned to the a-CA class. From the alignment showed in Figure 2, it clearly appears that some of the crucial hallmarks of the a-CAs are not present in the PfCA sequence. Indeed, for the catalytic mechanism of a-CAs several amino acid residues (other than the zinc ligands) are crucial and rather conserved in such enzymes present in very diverse kingdoms, from bacteria to protozoa,


3


5′ ATGAAGCTTTTATATTTACTATATCCCATTTTACTCTTTTACAACGTAAACGTATTTATTAACTATAAGAAGAGTAGACTAATGCTTGAAATGATAGATA M

K

L

L

Y

L

L

Y

P

I

L

L

F

Y

N

V

N

V

F

I

N

Y

K

K

S

R

L

M

L

E M

I

D

K

AATATAATACCCATTTTGTACAAACAACCAAACCTTATTACGAATTTAATGTAACTAATCTAACTAATTCCAAAAAAAAAAAAAAAAAAAAAAAAAGGGA Y

N

T

H

F

V

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T

T

K

P

Y

Y

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F

N

V

T

N

L

T

N

S

K

K

K

K

K

K

K

K

R

E

AAATCACCTGATCGGTTCAGGTGAAAATATGCAAAAAAAGGATGAAAAAAATATAAAGGATTTTCATATAAATGATTATGAAATAGATGGGAAAACAATT N

H

L

I

G

S

G

E

N

M

Q

K

K

D

E

K

N

I

K

D

F

H

I

N

D

Y

E

I

D

G K

T

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CATAATAAAGAGAACAAGGATTCCTTTAAAATGAATAAAAATAAATTAAACGATAACGAAGAATTATTTTATATGGACAATATATTATCTTATAAACCAA H

N

K

E

N

K

D

S

F

K

M

N

K

N

K

L

N

D

N

E

E

L

F

Y

M

D

N

I

L

S Y K P N

ATAAAAAGAAGTTGTTTACTTATTCCTTTTCCGAAAATGAAGGAAATTCTGAAAAAGAAGAAACCCTTTATAATTTTAAAAATATGAAAAATATAAATAG


K

K K

L

F

T

Y

S

F

S

E

N

E

G

N

S

E

K E

E

T

L

Y

N

F

K

N

M

K

N I

N

S

CGTACAAAATAATATTAACAAGACCTTTTTATATAACAAATTGAAAAATGTAGATTATTATGAGCATGGTTATAATTGGGATATAGGTCAATGTAAAACA V

Q

N

N

I

N

K

T

F

L

Y

N

K

L

K

N

V

D

Y

Y

E

H

G

Y

N

W

D

I

G

Q C

K

T

GGGAAATATCAATCTCCTGTTGATTTACCTATGAAAGATTTAAAGGAGAGAGAATTAAAAAATATAAGTGATGTGTATTTAAATTTATTTGACGATGACA G

K

Y

Q

S

P

V

D

L

P

M

K

D

L

K

E

R

E

L

K

N

I

S

D

V

Y

L

N

L

F D D D N

ATTATGCATGGAACAATTATAACAAACCATGGATGAAAGGAGATTTTTTTTATTATTATGAATATTTTATAAAAAAAATTGTTATTAATAGACAAAATAA Y

A

W

N

N

Y

N

K

P

W

M

K

G

D

F

F

Y

Y Y

E

Y

F

I

K

K

I

V

I

N

R Q

N

N

TATATTTCAAATAAAAGCTGCAAGAGATGGAATAATACCATTTGGTGTGTTATTTACTACTGAACAACCTGCTATGTTTTATGCAGATCAAATCCATTTT I

F

Q

I

K A

A

R

D

G

I

I

P

F

G

V

L

F

T

T

E

Q

P

A

M

F

Y

A

D

Q I H F

CATGCTCCTAGCGAACATACATTTCAAGGTTCAGGTAATAGAAGAGAAATTGAAATGCAAATATTTCATAGTACAAATTATTTTTATGATATACAAGATG H A

P

S

E

H

T

F

Q

G

S

G

N

R

R

E

I

E

M

Q

I

F H S

T

N

Y

F

Y

D I

Q

D

D

ATAAATCCAAATATAAAAAAAAATACGGGTTACATATATATAATAATTTAAAAAAAAATTCAAAAGAAACTTCAAAAAAAGATTCAAGTAGATATCATTC K

S

K

Y

K

K K

Y

G

L

H

I

Y

N

N

L

K

K

N

S

K

E

T

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K

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D

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S

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Y

H

S

TTATCTTATGTCCTTTCTAATGAATAGCTTATCAAATGAACAATTACAAAACAAATATAATAAAAAAAAAAGAATAAAAAAGATGAAAAACCAATATGAA Y

L

M

S

F

L

M

N

S

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S

N

E

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L

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Y

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M

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GTAATATCTATTACATTTACTAGTGCAGAAATTAATGCTTCAACTATTAATGCTTTTAAGAAATTACCATCAGAAAAATTTCTAAGAACTATAATAAATG V

I

S

I

T

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A

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A

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K

K

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T

I

I

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V

TATCAAGTGCAGTTCACGTCGGCTCAGATCCAACCTTGGTGGAATTAAAGGACGCTTTAAACCTGGATGCCTTGATGATGATGTTAAATATTGAAGACAT S

S

A

V

H

V

G

S

D P T L G N K

V

E

L

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D

A

L

N

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M

M

L

N

I

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D

M

GCAATTTTTGTCATATCAAGGATCTTCTACATTACCCCTATGTGATGAGAATGTATCCTGGAAAGTAGCCAAACAACCTTTGCCTGTATCAACTGAAACC Q

F

L

S

Y

Q

G

S

S

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N

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ATTTTAAATTTTTATTATCTCCTAAAAAAACATACACCTAATTATTCAGGTAGCGATAATGATAATTACAGGAGTTTACAAAATGTAGAAGATAATACAA I

L

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Y

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L

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K

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T

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GACATTATAGAAAGTTTTCTTTAGTTCAAGTTTTTCCTATACAAGTATTAATTTCATCAGCTATATCAAATATAGAGGATAAGAAAGTTATTAATATTAT H

Y

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TAAAGATATATCTCCTAAAAATATGTCCTTCACATATTATTCTAAATGGGATATATATTTTATTTTATTTATCTTTTATAACATTGTATTGTTCTTATTT K

D

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S

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TGA * 3′

Figure 1. Nucleotide and translated amino acid sequences of Plasmodium falciparum CA (Pf3D7_plasmodium). In grey, the amino acid sequence of the truncated malarial enzyme (PfCA) expressed in our laboratories [7]. The sequence contains three additional C-terminal amino acids not present in the native form (DPT changed to GNK). The putative zinc ion ligands are indicated by bold letters.

4




Figure 2. Alignment of the amino acid sequences of the truncated P. falciparum CA (PfCA), with the human a-CA isoforms I (hCA). . To perform the alignment six ‘missing’ residues have to be inserted in the protozoan enzyme sequence, and five in the mammalian enzymes sequences to align the Zn(II) ion ligands (in red). The proton shuttle residue His64 (in blue) and the gatekeeper residues (Glu106 and Thr199, in orange) are the residues crucial for the catalytic activity of the a-CAs. The hCA I numbering system has been used.

0.82 0.527 0.929

STPCA_coral OosCA_ncmatodc NgaCA_alga CAH-4a_nematode CAH-4b_ncmatodc

1 0.919 0.244

0.277

0.12 0.915

HumCAII_human HumCAI_human STPCA-2_coral TspCA_nematode AaeCA_mosquito DmeCA_fly

Preich_plasmodium

1

0 Pf3D7_plasmodium

PfIT_plasmodium 1 Py17X_plasmodium

1 0.929

0.355 Py17XNL_plasmodium

Pcchab_plasmodium

0.869

0.547

0.925

0.06

0.344 0.911 0.537 0.374 0.892

1 Pvvinc_plasmodium 0.983

Pvpet_plasmodium PbANKA_plasmodium SmaCA_fungus AthCA_plant Tcru_bacterium SspCA_bacterium Hpy1CA_bacterium Ssa1CA_bacterium NgonCA_bacterium CreCA_alga

Figure 3. Bootstrap majority rule consensus tree of Plasmodia CAs sequences and a-CAs from different organisms (nematode, coral, plant fungus, alga, fly and human CAs). The tree was constructed using the program PhyML 3.0 [54,55]. Bootstrap values on 100 replicates are reported at branch points. Class, organisms, accession numbers and cryptonyms of the sequences used in the alignment have been indicated in Table 2.

plants, and animals. Among them are the proton shuttle residue (His64 in most such enzymes) and the gatekeeper residues, Glu106 and Thr199, and they are not conserved in

the protozoan enzyme PfCA (Figure 2). The analysis of the hallmarks which we performed here clearly differentiate the h-CA sequences of Plasmodia or other protozoans, from


5


N CH3CONH

H3C

N

N SO2NH2

S

CH3CON

N

N SO2NH2

S

AAZ

EZA NHEt

NHEt SO2NH2

SO2NH2

Me O

MeO(CH2)3

O

DCP


S

S

Cl

SO2NH2

N

N

S

N H

O

S

S

O

O

DZA

O

SO2NH2

S

MZA

SO2NH2

Cl

EtO

BRZ

N

O NH2 O S O O

O

SO2NH2

SO2NH2

S

O

O

N

O

BZA

O

TPM

ZNS

OCH3 O N H

H N N

O

O S

Cl

N H SO2NH2

SO2NH2

SLP

IND SO2NH2

SO2NH2

SO2NH2

H N Me N N

Me O

N

VLX

N

Cl

O S O

HN O

SO2NH2

S O

F3C

CLX

SLT

HCT

Chart 2. Clinically used sulfonamides/sulfamates AAZ-HCT.

those of the a-CAs present in many species all over the phylogenetic tree (including some protozoa, such as for example T. cruzi [21]).

Phylogenetic analysis of PfCA and related enzymes from Plasmodia 3.

We have constructed phylogenetic trees to better investigate the relationship of the Plasmodia CA sequences with those of a-CAs from different organisms, such as nematodes, corals, plants, fungi, algae, flies and humans. From the dendrogram shown in Figure 3, the Plasmodia CAs appeared closely associated with each other, and clustered in a distinct branch from 6

the a-class enzymes. The Plasmodia CAs branch was near the clusters containing the plant, fungal, bacterial and algal CAs considered in the present analysis (Figure 3). To explore the relationship of the h-CAs with the other classes of enzymes, such as the b-, g-, d- and z-CAs, we have constructed a phylogenetic tree including in the analysis all the aforementioned classes (Figure 4). Figure 4 showed that Plasmodia CAs formed a distinct cluster phylogenetically distant from those containing the b-and z-CAs, whereas they were more correlated to the a-, g- and d-CA families. The plasmodia CAs considered in the present paper are thus difficult to classify. On the one hand, they have some features typical of a-CAs, but in other aspect such as the proton shuttle



TpsCA_ζ MpuCA_ζ

0.954 0.992

TweCA_ζ AbaCA_β

0.956

0.605

0.865

PgiCA_β MinCA_β Cab_β

0.879 0.999 0.568 0.827

ZmaCA_β VraCA_β 0.978 0.584 FbiCA_β AthCA_β HpyCA_β LpnCA_β

0.945 0.305

EcoCA_β


0.76

SpoCA_β 0.981 BsuCA_β 0.931 BthCA_β 0.86 0.959 CspCA_β 0.973 CreCA_β SceCA_β

0.591

DbrCA_β 0.75 1

LpoCA_δ TweCA_δ Bpr_δ

CreCA_γ PgiCA_γ AthCA_γ CAM_γ

0.714 0.871 0.973 0.605 0.967

PseCA_γ BglCA_γ

0.956 0.999

Preich_plasmodium

0 Pf3D7_plasmodium PfIT_plasmodium 0.944 1 Py17X_plasmodium 0.606Py17XNL_plasmodium

0.633

0.95

1 Pcchab_plasmodium 0.717 Pvvinc_plasmodium 0.988 Pvpet_plasmodium PbANKA_plasmodium HpylCA_α SspCA_α

0.954 0.329 0.61

HumCAII_α

1

HumCAI_α NgonCA_α

Figure 4. Phylogenetic tree constructed using amino acid sequences of the Plasmodia CAs and the known a-, b-, g-, d-, z-CA classes. The tree was obtained with the program PhyML 3.0. Bootstrap values on 100 replicates are reported at branch points. Class, organisms, accession numbers and cryptonyms of the sequences used in the alignment have been indicated in Table 2.

residue and the gatekeeper residues Thr199 they do not appear to be closely related to the a-CAs (Figure 3). Possibly, the CAs found in Plasmodia may be the results of modifications of an ancestral a-CA gene, which originated a new genetic family, the h-CAs [7].

4.

Kinetic properties of PfCA

The truncated form of the enzyme, PfCA, showed catalytic activity typical of a CA, having the following kinetic properties for the CO2 hydration reaction to bicarbonate and protons: kcat


7



Table 1. Kinetic parameters for the CO2 hydration reaction catalyzed by various CAs belonging to the various families. Isozyme

Class

Organism

kcat(s-1)

hCAI hCAII Can2 FbiCA1 PgiCA CdCA1-R1 ZnCA1-R1 TweCA Plasmodium falciparum CA

a a b b g z z d h

H. sapiens H. sapiens Cryptococcus neoformans Flaveria bidentis Porphyromonas gingivalis T. weissflogii T. weissflogii T. weissflogii P. falciparum

2.0 1.4 3.9 1.2 4.1 1.5 1.4 1.3 1.4

105 106 105 105 105 106 106 105 105

kcat/Km(M-1.s-1) 5.0 1.5 4.3 7.5 5.4 1.4 1.6 3.3 5.4

107 108 107 106 107 108 108 107 106

KI (acetazolamide) (nM) 250 12 10.5 27 324 82 58 83 170

The a-class CAs were the human cytosolic isozymes hCAI and II. The b-class includes the fungal enzyme Can2 from Cryptococcus neoformans and FbiCA1 from the plant Flaveria bidentis. The g-class enzyme was PgiCA from the anaerobic bacterium Porphyromonas gingivalis,19 whereas the d and z-class enzymes (the last with zinc and cadmium at the active site) were from the diatom Thalassiosira weissflogii. Inhibition data with the clinically used sulfonamide acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide) are also provided [7]. CA: Carbonic anhydrase.

of 1.4 105 s-1 and kcat/Km of 5.4 106 M-1 s-1 [7]. Table 1 shows the kinetic parameters for the same reaction catalyzed by CAs belonging to all six genetic families: the a-class CAs were the human cytosolic isozymes hCA I and II, the b-class included the fungal enzyme Can2 from Cryptococcus neoformans and FbiCA1 from the plant Flaveria bidentis; the g-class enzyme was PgiCA from the anaerobic bacterium Porphyromonas gingivalis, whereas the d- and z-class enzymes (the last with zinc and cadmium at the active site) were from the diatom Thalassiosira weissflogii. It may be observed that PfCA has a kinetic constant kcat in the same range with the slow human isoform hCAI or the d-class enzyme TweCA, whereas the kcat/Km value of the h-CA was similar to the plant b-CA FbiCA1. Inhibition data with the clinically used sulfonamide acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide) are also shown in Table 1, proving that all six CA classes are inhibited by this compound with variable efficacy. hCAII and FbiCA1 were the most sensitive enzymes to this sulfonamide inhibitor (KIs of 12 -- 27 nM), whereas hCA I, PgiCA and PfCA were the least inhibited enzymes, with KIs ranging between 170 and 324 nM (Table 2). These data at least demonstrate two important things: PfCA has a relevant catalytic activity for the physiologic reaction, and it has an inhibition profile with AAZ distinct from other classes of such enzymes. 5.

PfCA inhibition studies

The first inhibition study of PfCA was done with inorganic and complex anions as well as other molecules known to interact with CAs, such as sulfamide, sulfamic acid, and phenylboronic/arsonic acids [7], and allowed the identification of several low micromolar h-CAIs (Table 3). It may be observed that both complexing as well as non-complexing inorganic anions such as halides, cyanide, azide, bicarbonate, trithiocarbonate, dithiocarbamate, and so on, are weak, millimolar 8

h-CAIs, although some of them are much more efficient inhibitors of the a-class enzymes from H. sapiens and the protozoan T. cruzi. However sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid showed low micromolar affinity for PfCA, with inhibition constants ranging between 6 and 9 µM (Table 3). As sulfonamides and sulfamates represent the main class of CAIs, a second inhibition study [53] of the h-CA from P. falciparum took into consideration these agents (Chart 2), many of which are in clinical use for decades for the treatment and prevention of a variety of disorders, from glaucoma and edema, to epilepsy, obesity, and so on. [48]. The inhibition data of the human isoforms hCA I and hCA II, of the protozoan ones from T. cruzi (TcCA), Leishmania donovani chagasi (LdcCA) and P. falciparum (PfCA) with these clinically used drugs AAZ -- HCT are shown in Table 4. It may be observed that all sulfonamides/sulfamates investigated so far showed inhibitory activity, although no compound with a KI < 100 nM was detected so far for the h-class enzyme. The best inhibitors were ethoxzolamide EZA and sulthiame SLT, with KIs of 131 -- 132 nM, followed by acetazolamide AAZ, methazolamide MZA, and hydrochlorothiazide HCT (KIs ranging from 153 to 198 nM, Table 4). The striking observation is that these rather efficient CAIs belong to a variety of scaffolds and chemical classes, ranging from aromatic primary sulfonamides (SLT) to monocyclic heterocyclic derivatives (AAZ, MZA) and bicyclic such derivatives (EZA, HCT). This is a very interesting observation which leads us to speculate that it should be possible to develop low nanomolar CAIs targeting h-class CAs starting form one of these many lead compounds. A range of other drugs, such as brinzolamide BRZ, topiramate TPM, zonisamide ZNS, indisulam IND, valdecoxib VLX and celecoxib CLX, also showed significant inhibitory action against PfCA, with KIs ranging from 217 to 308 nM,




Table 2. CA class, organism, accession number and cryptonym of the sequences used in primary sequences alignment and in the phylogenetic analysis. CA class

Organism

Accession number

Cryptonym

Alpha (a)

Helicobacter pylori J99 Homo sapiens, isoform II Homo sapiens, isoform I Sulfurihydrogenibium yellowstonense YO3AOP1 Streptococcus salivarius Thiomicrospira crunogena Neisseria gonorrhoeae Ostertagia ostertagi Caenorhabditis elegans Caenorhabditis elegans Trichinella spiralis Stylophora pistillata Stylophora pistillata Aedes aegypti Drosophila melanogaster Nannochloropsis gaditana Chlamydomonas reinhardtii Sordaria macrospora k-hell Arabidopsis thaliana Schizosaccharomyces pombe Brucella suis 1330 Burkholderia thailandensis Coccomyxa sp. Chlamydomonas reinhardtii Acinetobacter baumannii Porphyromonas gingivalis Myroides injenensis Zea mays Vigna radiata Flaveria bidentis, isoform I Arabidopsis thaliana Helicobacter pylori Legionella pneumophila Escherichia coli Methanobacterium thermoautotrophicum Saccharomyces cerevisiae Dekkera bruxellensis Pseudomonas sp. Burkholderia gladioli Methanosarcina thermophila Chlamydomonas reinhardtii Arabidopsis thaliana P. gingivalis Thalassiosira weissflogii Thalassiosira pseudonana Emiliania huxleyi Bathycoccus prasinos Lingulodinium polyedrum Thalassiosira weissflogii

NP_223829.1 AAH11949.1 NP_001158302.1 ACD66216.1

HpylCA_a or HpylCA_bacterium HumCAII_a or HumCAII_human HumCAI_a or HumCAI_human SspCA_a or SspCA_bacterium

WP_002888224.1 WP_011370966.1 CAA72038.1 AAT66640.1 NP_510265.1 NP_510264.1 XP_003369705.1 ACA53457.1 ACE95141.1 AAL72625.1 NP_648555.1 EWM20524.1 XP_001692290.1 XP_003347723.1 NP_850685.1 CAA21790 NP_699962.1 ZP_02386321 AAC33484.1 XP_001699151.1 YP_002326524 YP_001929649.1 ZP_10784819 NP_001147846.1 AAD27876 AAA86939.2 AAA50156 BAF34127.1 YP_003619232 ACI70660 GI:13786688 GAA26059 EIF49256 ZP_10427314.1 YP_004359911.1 ACQ57353.1 XP_001703237.1 NP_564091.1 YP_001929649.1 AAV39532.1 XP_002287620.1 ABG37687.1 CCO20234.1 ABS87870.1 AAX08632.1

Micromonas pusilla Thalassiosira pseudonana Plasmodium reichenowi Plasmodium falciparum 3D7 P. berghei ANKA P. falciparum Vietnam Oak-Knoll P. yoelii 17X

XP_003063214.1 XP_002287620.1 CDO65199.1 PF3D7_1140000 PBANKA_090900 ETW15286.1 ETB57522.1

SsalCA_bacterium Tcru_bacterium NgonCA_a or NgonCA_bacterium OosCA_nematode CAH-4a_nematode CAH-4b_nematode TspCA_nematode STPCA_coral STPCA-2_coral AaeCA_mosquito DmeCA_fly NgaCA_alga CreCA_alga SmaCA_fungus AthCA_plant SpoCA_b BsuCA_b BthCA_b CspCA_b CreCA_b AbaCA_b PgiCA_b MinCA_b ZmaCA_b VraCA_b FbiCA_b AthCA_b HpyCA_b LpnCA_b EcoCa_b Cab_b SceCA_b DbrCA_b PseCA_g BglCA_g CAM_g CreCA_g AthCA_g PgiCA_g TweCA_delta TpsCA_delta EhuCA_delta Bpr_delta LpoCA_delta TweCA_zeta, CDCA1 MpuCA_zeta TpsCA_zeta Preich_plasmodium Pf3D7_plasmodium PbANKA_plasmodium PfIT_plasmodium Py17X_plasmodium

Beta (b)

Gamma (g)

Delta (d)

Zeta (z)

Eta (h)

CA: Carbonic anhydrase.


9


Table 2. CA class, organism, accession number and cryptonym of the sequences used in primary sequences alignment and in the phylogenetic analysis (continued). CA class

Organism

Accession number

Cryptonym

P. yoelii yoelii 17XNL P. vinckei vinckei P. vinckei petteri P.chabaudi chabaudi P. falciparum CA (residues 211 -- 445) [11,12,16,17]

XP_726574.1 KEG02328.1 EUD73019.1 PCHAS_071030 -

Py17XNL_plasmodium Pvinc_plasmodium Pvpet_plasmodium Pcchab_plasmodium Plasmodium falciparum CA


CA: Carbonic anhydrase.

Table 3. Inhibition constants of anion inhibitors against a-CAs from mammals (hCAI, and II, human isoforms, and the protozoan enzyme from Trypanosoma cruzi, TcCA) and the h-CA PfCA from Plasmodium falciparum for the CO2 hydration reaction, at 20 C and pH 7.5 [7]. Inhibitor

-

F ClCNOSCNCNN3HCO3SeCNCS32Et2NCS2FSO3NH(SO3)22H2NSO2NH2 H2NSO3H Ph-B(OH)2 Ph-AsO3H2

hCAI

hCAII

TcCA

PfCA

> 300 6 0.0007 0.2 0.0005 0.0012 12 0.085 0.0087 0.00079 0.79 0.31 0.31 0.021 58.6 31.7

> 300 200 0.03 1.60 0.02 1.51 85 0.086 0.0088 0.0031 0.46 0.76 1.13 0.39 23.1 49.2

0.90 0.81 0.080 0.084 1.01 1.12 0.58 0.78 0.093 0.005 15.8 7.1 0.12 10.6 0.86 0.62

5.78 9.76 0.81 0.95 0.76 5.60 0.78 0.87 4.36 0.55 9.45 5.96 0.008 0.009 0.007 0.006

being thus considered medium potency inhibitors. Again they belong to very heterogeneous classes, proving that PfCA is a druggable target. The least effective PfCA inhibitors were dichlorophenamide DCP, dorzolamide DZA, benzolamide BZA, and sulpiride SLP, with KIs ranging from 542 to 1170 nM (Table 4). From the data of Table 4 it should also be observed that the a-class enzymes of human or protozoan origin (hCA I, II and TcCA, respectively) as well the protozoan b-CA from L. donovani chagasi showed very different inhibition profiles with these sulfonamides/sulfamates, compared to the h-CA from P. falciparum (Table 4).

Expert opinion

Malaria is a serious medical problem, with the emergence of strains resistant to many antimalarial drugs worldwide, and 10

Inhibitor/Enzyme class

KI [mM]

CA: Carbonic anhydrase; pfCA: Plasmodium falciparum carbonic anhydrase.

6.

Table 4. Inhibition of human isoforms hCA I and hCA II, of the protozoan ones from T. cruzi (TcCA), Leishmania donovani chagasi and Plasmodium falciparum (PfCA) with the clinically used drugs AAZ -- HCT.

AAZ MZA EZA DCP DZA BRZ BZA TPM ZNS SLP IND VLX CLX SLT HCT

KI* (nM) hCAI a

‡

250 50 25 1200 50000 45000 15 250 56 1200 31 54000 50000 374 328

hCAII a 12 14 8 38 9 3 9 10 35 40 15 43 21 9 290

‡

TcCA§ LdcCA{ a b

PfCA# h

61.6 74.9 88.2 128 92.9 87.3 93.6 85.5 867 87.9 84.5 82.7 91.1 71.9 134

170 198 131 542 963 260 1330 295 246 1170 308 226 217 132 153

91.7 87.1 51.5 189 806 764 236 > 100,000 > 100,000 > 100,000 316 338 705 834 50.2

* Errors in the range of 5 -- 10% of the shown data, from 3 different assays. z Human recombinant isozymes, stopped flow CO2 hydrase assay method, from [48]. § Recombinant protozoan enzyme, stopped flow CO2 hydrase assay method, from [21]. { Recombinant protozoan enzyme, stopped flow CO2 hydrase assay method, from [22]. # Recombinant protozoan enzyme, stopped flow CO2 hydrase assay method, from [55]. CA: Carbonic anhydrase; pfCA: Plasmodium falciparum carbonic anhydrase.

the expansion of the disease to new areas due to the global climatic changes. Most antimalarials were discovered decades ago, with no new therapeutic approaches available in the recent period, which makes the problem even more challenging. The h-class CA was recently discovered as the sixth genetic family of this superfamily of metalloenzymes, being present in only in some protozoa, among which various Plasmodium species, which are the malaria-provoking pathogens. Recent, a detailed biochemical, kinetic and phylogenetic analysis afforded a clear insight regarding the differences between




this new class, the h-CA and the other CA families (the a-, b, g-, d- and z-CAs). Like a-, g- and d-class enzymes, the hCAs have the catalytic Zn(II) ion coordinated by three histidine residues and a water molecule, which acts as a nucleophile in the conversion of CO2 to bicarbonate and protons. h-CAs seem to be more closely related to the a-CAs, but there are notable differences between the two classes of enzymes, such as the sequence of the three histidine residues coordinating the metal ion within the enzyme active site (x, x + 2, x + 24 for the h-CAs, and x, x + 2, x + 25 for the a-CA); the lack of the proton shuttle residue (His64) and gatekeeper residues, Glu106 and Thr199 in the h-CAs, which are conserved in all a-CAs investigated so far. No X-ray crystal structures or models of h-CAs are available so far, but these enzymes also have a much larger number of amino acid residues (600 amino acid polypeptide chain) compared to the a-CAs (typically 260 -- 280 amino acid residues polypeptide chain). The kinetic properties of a truncated P. falciparum h-CA (PfCA) were measured by a stopped-flow technique, showing the enzyme to possess a moderate but significant activity for the CO2 hydration reaction to bicarbonate and protons, with a kcat of 1.4 x 105s-1 and a kcat/Km of 5.4 106 M-1 s-1. Several inhibition studies with anions and sulfonamides/ sulfamates, allowed the identification of interesting lead compounds, which may be useful for developing specific inhibitors for the h-CAs. Indeed, the small inorganic complexing and non-complexing anions were not effective inhibitors (KIs in the millimolar range), whereas sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid showed low micromolar affinity for PfCA, with inhibition constants ranging between 6 and 9 µM. Although all sulfonamides/sulfamates investigated so far showed inhibitory activity, although no compound with a KI < 100 nM was detected so far for the h-class enzyme. The best inhibitors identified to date were ethoxzolamide and sulthiame, with KIs of 131 -- 132 nM,

followed by acetazolamide, methazolamide and hydrochlorothiazide. A number of other such compounds (e.g., brinzolamide, topiramate, zonisamide, indisulam, valdecoxib and celecoxib, also showed significant inhibitory action against PfCA, with KIs ranging from 217 to 308 nM). A striking observation was that the efficient PfCA inhibitors detected so far belong to a variety of scaffolds and chemical classes, ranging from aromatic primary sulfonamides to monocyclic/bicyclic heterocyclic derivatives and compounds with a complicated scaffold such as the sugar derivative topiramate or the coxibs, celecoxib and valdecoxib. Considering that in literature there are few inhibition studies for the h-CAs and that these enzymes are druggable targets, it is important to detect soon low nanomolar inhibitors for this new class of CAs. As relevant drug resistance problems emerged against most antimalarials in clinical use, the discovery of h-CA-specific inhibitors may lead to novel such agents with a new mechanism of action. However this research is at this moment in a very preliminary phase, with the target just identified and characterized. It should however be mentioned that Krungkrai’s group reported [11,12] effective inhibition of growth of the malarial parasite in vitro and in an animal model of the disease, by using some sulfonamides tested as inhibitors of the presumed a-CA from the malaria parasite. Those compounds were not yet evaluated for the inhibition of h-CAs, and probably the earlier studies should be reevaluated by considering this new drug target.

Declaration of interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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Affiliation

Claudiu T Supuran1,2 & Clemente Capasso†3 † Author for correspondence 1 Universita degli Studi di Firenze, Dipartmento di Chimica Ugo Schiff, Via della Lastruccia 3, Rm. 188, 50019 - Sesto Fiorentino (Florence), Italy 2 Universita degli Studi di Firenze, Neurofarba Deptartment, Section of Pharmaceutical and Nutriceutical Sciences, Via U. Schiff 6, 50019 Sesto Fiorentino (Florence), Italy 3 Istituto di Bioscienze e Biorisorse (IBBR)- CNR, Via P. Castellino 111, 80131, Napoli, Italy E-mail: [email protected]

13

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Evaluation of spiropiperidine hydantoins as a novel class of antimalarial agents.

Characterisation of carbonic anhydrases from tissues of the cat.

2,4-Diaminothienopyrimidines as orally active antimalarial agents.

Serine Proteases of Malaria Parasite Plasmodium falciparum: Potential as Antimalarial Drug Targets.

Molecular and biochemical characterization of carbonic anhydrases of Paracoccidioides.

Drug discovery studies on quinoline-based derivatives as potential antimalarial agents.

Natural product polyamines that inhibit human carbonic anhydrases.

Sulfonamide inhibition study of the carbonic anhydrases from the bacterial pathogen Porphyromonas gingivalis: the β-class (PgiCAb) versus the γ-class (PgiCA) enzymes.

Fluorescent Silica Nanoparticles with Multivalent Inhibitory Effects towards Carbonic Anhydrases.

Role of carbonic anhydrases in skin wound healing.

Fluorescent Silica Nanoparticles with Multivalent Inhibitory Effects towards Carbonic Anhydrases.