Sulfonamide inhibition studies of the η-class carbonic anhydrase from the malaria pathogen Plasmodium falciparum

Accepted Manuscript Sulfonamide inhibition studies of the η-class carbonic anhydrase from the malaria pathogen Plasmodium falciparum Daniela Vullo, Sonia Del Prete, Gillian M. Fisher, Katherine T. Andrews, SallyAnn Poulsen, Clemente Capasso, Claudiu T. Supuran PII: DOI: Reference:

S0968-0896(14)00847-5 http://dx.doi.org/10.1016/j.bmc.2014.12.009 BMC 11941

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

29 October 2014 1 December 2014 4 December 2014

Please cite this article as: Vullo, D., Prete, S.D., Fisher, G.M., Andrews, K.T., Poulsen, S-A., Capasso, C., Supuran, C.T., Sulfonamide inhibition studies of the η-class carbonic anhydrase from the malaria pathogen Plasmodium falciparum, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.12.009

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Sulfonamide inhibition studies of the η-class carbonic anhydrase from the malaria pathogen Plasmodium falciparum Daniela Vullo,a Sonia Del Prete,b Gillian M. Fisher,c Katherine T. Andrews,c Sally-Ann Poulsen,c Clemente Capasso,b* and Claudiu T. Supurana,d* a

Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via

della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy. b

Istituto di Bioscienze e Biorisorse (IBBR) – CNR, Via P. Castellino 111, 80131 Napoli, Italy.

c

Eskitis Institute for Drug Discovery, Griffith University, Nathan, Queensland 4111, Australia.

d

Università degli Studi di Firenze, Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche, Polo

Scientifico, Sesto Fiorentino, Firenze, Italy.

Abstract: The η-carbonic anhydrases (CAs, EC 4.2.1.1) were recently discovered as the sixth genetic class of this metalloenzyme superfamily, and are so far known only in protozoa, including various Plasmodium species, the causative agents of malaria. We report here an inhibition study of the η-CA from P. falciparum (PfCA) against a panel of sulfonamides and one sulfamate compound, some of which are clinically used. The strongest inhibitors identified were ethoxzolamide and sulthiame, with KIs of 131-132 nM, followed by acetazolamide, methazolamide and hydrochlorothiazide (KIs of 153-198 nM). Brinzolamide, topiramate, zonisamide, indisulam, valdecoxib and celecoxib also showed significant inhibitory action against PfCA, with KIs ranging from 217 to 308 nM. An interesting observation was that the more efficient PfCA inhibitors are representative of several scaffolds and chemical classes, including benzene sulfonamides, monocyclic/bicyclic heterocyclic sulfonamides and compounds with a more complex scaffold (i.e., the sugar sulfamate derivative, topiramate, and the coxibs, celecoxib and valdecoxib). A comprehensive inhibition study of small molecules for η-CAs is needed as a first step towards assessing PfCA as a druggable target. The present work identifies the first known η-CA inhibitors and provides a platform for the development of next generation novel PfCA inhibitors. Keywords: carbonic anhydrase; η-CA-class enzyme; inhibitor; sulfonamide; sulfamate; Plasmodium falciparum _____ *Corresponding

authors:

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1. Introduction Malaria, a mosquito-borne disease of humans and other animal species, is caused by parasitic protozoa species belonging to the genus Plasmodium. Six different Plasmodium species infect humans: P. falciparum, P. vivax, P. ovale (two species), P. malariae and the zoonotic P. knowlesi. Globally malaria afflicts >200 million people and kills >600,000 annually, mainly young children in sub-Saharan Africa, with most deaths caused by P. falciparum infection.1-4 Malaria parasites follow a complex lifecycle that involves an intermediate host such as humans and the definitive host, the mosquito vector. Following injection of sporozoite stage parasites from an infected female Anophelene mosquito into a human host, Plasmodium parasites move to the liver and invade hepatocytes where they replicate to form merozoites that are ultimately released into the blood circulation. Plasmodium merozoites can then invade erythrocytes and undergo cycles of asexual replication within these cells, resulting in the clinical symptoms of malaria. During this part of the lifecycle, sexual stage gametocytes can also form, and when taken up by a feeding female Anophelene mosquito, can undergo sexual reproduction in the mid-gut of the mosquito. This ultimately results in the completion of the life cycle through formation of sporozoites that can then be transferred to another individual by the mosquito vector during a blood meal.5,6 Although effective drugs have been available for many years, drug resistant Plasmodium parasites have emerged to all clinically used antimalarials, making the treatment and prevention of the disease a very challenging medical issue.3,4,7,8 Even the gold-standard artemisinin combination therapies (ACTs), drugs that have contributed to decreases in malaria mortality over the past decade are now reported in several regions as being less effective due to possible drug resistance.4d The clinically used antimalarials such as the natural product quinine, the widely used synthetic drugs chloroquine and its analogs (amodiaquine and primaquine), proguanil, the sulfa drugs sulfadoxine and pyrimethamine were developed in the ‘50s and ‘60s and, since the introduction of artemisinin, no new antimalarial chemotype has been introduced since the late 1990’s.7,8 Despite promising recent progress on a vaccine which is targeted to children in malaria endemic areas,8f clinical approval has not yet been obtained and even when approved this vaccine may not be broadly applicable as it is only partially effective and has not yet been assessed in the diverse groups susceptible to malaria.8g Thus, malaria prevention and treatment will continue to rely heavily on drugs that target one or more of the different parasite life cycle stages. To facilitate the development of new antimalarial drug leads, it is imperative that novel drug targets be explored in order to help prioritize those with potential downstream clinical potential.

Recently, our groups reported that Plasmodium species encode for a novel family of carbonic anhydrases (CAs, EC 4.2.1.1), the η-CAs,9 which in addition to the α-, β-, γ-, δ- and ζ-CA class enzymes are the sixth genetic family encoding such metalloenzymes reported to date.10,11 The CA enzymes catalyze a simple chemical reaction, the reversible inter-conversion between CO2 and bicarbonate, which generates a proton and bicarbonate (for the hydration reaction) or consumes one equivalent of protons (for the dehydration reaction). This makes these enzymes crucial for many processes connected with pH regulation, CO2 chemosensing, biosynthetic reactions employing CO2/bicarbonate as substrate, as well as other functions across organisms of diverse phylogeny.10-15 Like α-, γ- and δ-class enzymes, the η-CAs are predicted to have the catalytic Zn(II) ion coordinated by three histidine residues and the catalytic water molecule. The η-CA class appears closely related to the α-CAs, however they differ to the α-CAs by (i) a predicted sequence difference whereby the three histidine residues coordinating the metal ion within the enzyme active site for the η-CAs are positioned x, x+2, x+24, and for the α-CAs are positioned x, x+2, x+25 (where x represents the position of the first coordinating His residue in the amino acid sequence of the protein; for example for the human CAs, x = 94); and (ii) lack of the proton shuttle residue (His64) and gatekeeper residues Glu106 and Thr199, that are conserved in all characterized α-CAs.9 In addition, the η-CA enzymes identified to date have a much larger number of amino acid residues (600 amino acid polypeptide chain) compared to the α-CAs (typically 260-280 amino acid residues polypeptide chain).9 It has been previously proposed

8a,b

that Plasmodium CAs are involved in biosynthetic

reactions through which the parasite synthesizes purines and pyrimidines for DNA/RNA synthesis during its exponential growth and replication. Indeed, Plasmodium parasites synthesize pyrimidines de novo from HCO3-, adenosine-5’-triphosphate (ATP), glutamine (Gln), aspartate (Asp) and 5phosphoribosyl-1-triphosphate (PRPP), and the HCO3- used as substrate for the first enzyme involved in the pyrimidine pathway, is generated from CO2 through the action of CAs. 8a-c Although some earlier studies

8a,b

showed that sulfonamide-based CA inhibitors (CAIs)

interfere with the in vitro growth of P. falciparum, no systematic studies with this class of inhibitors were performed. P. falciparum CA (PfCA) was originally considered an α-class CA, and only very recently we showed that it belongs to a new genetic family, the η-CA class. In that study

9

we

showed that a truncated form of the P. falciparum enzyme PfCA showed catalytic activity typical of a CA with the following kinetic properties for the hydration reaction of CO2 to bicarbonate and protons: kcat of 1.4 x 105 s-1 and kcat/Km of 5.4 x 10 6 M-1 x s-1. We also observed that this activity was inhibited by the clinically used sulfonamide inhibitor acetazolamide (AAZ) with a KI of 170 nM.9 In addition, the PfCA inhibition properties of a large panel of inorganic anions and small molecules

(sulfamide, sulfamic acid) were investigated. No detailed studies of η-CA inhibition are known with more complex small molecules. Here we present the first study of η-CA inhibition against a panel of compounds comprising the sulfonamide and sulfamate zinc binding group, several of which are clinically used drugs. Krungkrai’s group 8a-c reported effective inhibition of growth of the malarial parasite in vitro and in an animal model of the disease using some sulfonamides tested as inhibitors of the presumed α-CA from the malaria parasite, however those compounds were not assayed for the inhibition of the endogenous CA catalyzed reaction.

2. Results and discussion The protozoa Trypanosoma cruzi encodes an α-class CA (TcCA) in its genome, while Leishmania donovani chagasi, another parasitic protozoa, encodes a β-class CA (LdcCA). TcCA is inhibited significantly by sulfonamides, thiols and anions,16 similarly LdcCA is inhibited by sulfonamides and thiols and some of these were effective in reduction of the growth of this parasite in vitro.17 We hypothesize that inhibition of protozoan CAs may lead to therapeutic agents that act with a novel mechanism of action. To help aid in testing this hypothesis, here we extend our knowledge of inhibition of pathogen CAs, specifically targeting the recently characterized Plasmodium enzyme PfCA. A selection of representative CA inhibitor scaffolds bearing sulfonamides and sulfamate functionality, compounds 1-24 as well as 15 clinically used or clinically tested agents, were investigated, Figure 1.18,19,20 The inhibition data against the human isoforms hCA I and hCA II, and protozoan CAs from T. cruzi (TcCA),16 L. donovani chagasi (LdcCA)17 and P. falciparum (PfCA) with the compounds 1-24 and the 15 clinically used/ tested drugs are provided in Table 1. Fig. 1 and Table 1 here All compounds showed CA inhibitory activity, with multiple inhibitors possessing KIs < 100 nM identified for the α-class TcCA and β-class LdcCA, however no compound with a KI < 100 nM was detected for the η-class PfCA. The best PfCA inhibitors were ethoxzolamide EZA and sulthuame SLT, with KIs of 131-132 nM, followed by acetazolamide AAZ, methazolamide MZA, and hydrochlorothiazide HCT (KIs ranging from 153 to 198 nM, Table 1). A striking observation is that these 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). While additional studies are required, this observation may provide chemical starting points for development of CAIs that target the η-class CAs. Of the remaining clinically used compounds brinzolamide BRZ, topiramate TPM, zonisamide ZNS, indisulam IND, valdecoxib VLX and celecoxib CLX showed moderate inhibition of PfCA, with KIs ranging from

217 to 308 nM. As for the stronger inhibitors, these moderate inhibitors similarly comprise heterogeneous structural motifs. The least effective PfCA inhibitors from the set of clinically tested compounds were dichlorophenamide DCP, dorzolamide DZA, benzolamide BZA and sulpiride SLP, with KIs ranging from 542 to 1170 nM (Table 1). Compounds 1-24 are simpler in structure than the clinically tested compounds and comprise either the benzene sulfonamide or thiadiazole sulfonamide scaffold decorated with small substituents on the aryl/heteroaryl ring or structures with two aromatic rings separated by variable spacer groups. Many of these simpler compounds showed effective, albeit moderate to weak inhibitory activity of PfCA. Compounds 1, 6, 12, 13, 16 and 17 had KIs in the range of 581-758 nM and are medium potency PfCA inhibitors. The remaining sulfonamides are weaker inhibitors, with KIs in the range of 3.65 – 15.0 µM (Table 1). In general compounds 1-24 are substantially weaker PfCA inhibitors than the clinically used compounds. Several structure-activity relationships (SAR) are apparent for these compounds. Firstly, halogen substitution of the sulfanilamide scaffold 2 (PfCA KI = 5.8 µM) either does not improve inhibition or is detrimental to inhibition (compare KI values of 2 with halogenated derivatives 7-9, 10 and 12). Secondly, the length of the alkyl spacer group present in compounds 5, 6 and 15-17 impacts on the PfCA inhibitory activity, inhibition improves from a methylene linker in 5 (PfCA KI = 3.6 µM) and 15 (PfCA KI = 4.5 µM) to the ethyl and propyl linkers of 6, 16 and 17 (PfCA KIs ~0.7 µM). Finally, the sulfanylated sulfanilamides 2124 showed very weak PfCA inhibition (PfCA KIs 8.4-15.0 µM). A full discussion of the SAR for these compounds against TcCA and LdcCA was previously described by ourselves,17,18 however the KI data is included here to allow the first comparison of protozoa CA inhibition across the α-, β- and η-CAs, Table 1. The inhibition profile is very different across the three enzyme classes, with the sulfonamides/sulfamates generally more effective inhibitors of the α- protozoa CAs than the P. falciparum η-CA, while for the and β- protozoa several compounds have good selectivity towards PfCA, such as 6 (>132-fold), TPM (>339-fold), ZNS (>406-fold) and SLP (>85-fold) This suggests that the active site architecture comprises differing attributes that will enable the development of compounds for selective targeting across these protozoa CAs. When comparing PfCA inhibition against the human counterparts present in human red blood cells, a foremost consideration in antimalarial drug discovery, some compounds stand out as having selectivity for PfCA versus hCA I. For example, VLX and CLX have >200-fold selectivity for PfCA (KI ~200 nM) versus hCA I (KI ~ 50 µM).

3. Conclusion

The η-class CA was recently discovered as the sixth genetic family of this superfamily of metalloenzymes,9 being present in only in some protozoa, including various Plasmodium species, the causative agent of malaria. Recently, a detailed biochemical, kinetic and phylogenetic analysis afforded a clear insight regarding the differences between this new class, η-CAs, and the other CA families (the α-, β-, γ-, δ- and ζ-CAs).9 The is the first PfCA inhibition study with small molecule sulfonamides and sulfamates and has identified a selection of compounds which may be useful starting points for developing more potent and specific inhibitors for the η-CAs in the future. All investigated compounds showed some degree of PfCA inhibitory activity although no compound with a KI < 100 nM was detected. The best inhibitors identified were ethoxzolamide EZA and sulthiame SLT, with KIs of 131-132 nM, followed by acetazolamide AAZ, methazolamide MZA, and hydrochlorothiazide HCT, with KIs ranging from 153 to 198 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 and heterocyclic derivatives. Considering the small number of inhibition studies reported at this moment for the ηCAs, these results demonstrate it is quite probable that effective, low nanomolar inhibitors may be developed. Given that drug resistance has emerged against most antimalarials in clinical use, the discovery of η-CA-specific inhibitors may lead to a novel therapeutic approach for malaria once the biology of PfCA has been further investigated in different life cycle stages.

4. Experimental Protocols 4.1. Chemistry. Sulfonamides 1-24 and AAZ-HCT were commercially available or reported earlier by us.21 All compounds were > 95 % purity, as assessed by HPLC. 4.2. CA activity measurements and inhibition studies. A stopped-flow CO2 hydration assay with an Applied Photophysics instrument has been used for measuring catalytic activity and inhibition of the PfCA.18 Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.4) as buffer and 20 mM NaClO4 for maintaining constant the ionic strength. The initial rates of the CA-catalyzed CO2 hydration reaction were followed for a period of 10-100 s.18 The concentrations of substrate (CO2) ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants, with at least six traces of the initial 5-10% of the reaction being used for determining the initial velocity, for each inhibitor. The uncatalyzed rates were determined subtracted from the total observed rates. Stock solutions of inhibitors (10 mM) were prepared in distilled-deionized water and dilutions up to 0.01

nM were done with the assay buffer. Enzyme and inhibitor solutions were preincubated prior to assay for 15 min (at room temperature), in order to allow for the formation of the E-I complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation as reported earlier by our groups. The kinetic parameters for the uninhibited enzymes were derived from Lineweaver-Burk plots, as reported earlier,9,22,23 and represent the mean from at least three different determinations. PfCA, a recombinant protein prepared as described earlier, 9 was used at 21.4 nM. Acknowledgments: This research was financed by an FP7 EU project (Dynano) to CTS, the Australian Research Council (FT0991213 to KTA, FT10100185 to SAP) and the Australian National Health and Medical Research Council (PhD Scholarship to GF).

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Table 1: Inhibition of human isoforms hCA I and hCA II, of the protozoan ones from T. cruzi (TcCA), L. donovani chagasi (LdcCA) and P. falciparum η-class enzyme (PfCA) with sulfonamides 1-24 and the clinically used drugs AAZ – HCT.18 Inhibitor/

KI* (nM) hCA Ia

Enzyme class α

hCA IIa

TcCAb

LdcCA c

PfCAd

α

α

β

η

1

28000

300

25460

5960

581

2

25000

240

57300

9251

5800

3

79

8

63800

8910

5885

4

78500

320

44200

> 100,000

5580

5

25000

170

7231

> 100,000

3650

6

21000

160

9238

> 100,000

758

7

8300

60

8130

15600

5545

8

9800

110

6925

9058

6175

9

6500

40

8520

8420

5440

10

7300

54

9433

9135

6310

11

5800

63

842

9083

12,450

12

8400

75

820

4819

4140

13

8600

60

534

584

618

14

9300

19

652

433

744

15

5500

80

73880

927

4490

16

9500

94

71850

389

704

17

21000

125

66750

227

726

18

164

46

84000

59.6

6780

19

109

33

810

> 100,000

5250

20

6

2

88.5

95.1

6705

21

69c

11 c

134

50.2

12800

22

164

46

365

136

15000

23

109

33

243

87.1

14600

24

95

30

192

73.4

8400

AAZ

250

12

61.6

91.7

170

MZA

50

14

74.9

87.1

198

EZA

25

8

88.2

51.5

131

DCP

1200

38

128

189

542

(Table 2, continued)

DZA

50000

9

92.9

806

963

BRZ

45000

3

87.3

764

260

BZA

15

9

93.6

236

1330

TPM

250

10

85.5

> 100,000

295

ZNS

56

35

867

> 100,000

246

SLP

1200

40

87.9

> 100,000

1170

IND

31

15

84.5

316

308

VLX

54000

43

82.7

338

226

CLX

50000

21

91.1

705

217

SLT

374

9

71.9

834

132

HCT

328

290

134

50.2

153

* Errors in the range of 5 – 10 % of the shown data, from 3 different assays. a Human recombinant isozymes, stopped flow CO2 hydrase assay method, from refs.10a,c b Recombinant protozoan enzyme, stopped flow CO2 hydrase assay method, from ref.16 c Recombinant bacterial enzyme, from ref.17 d Recombinant bacterial enzyme, this work.

SO2NH2

SO2NH2

SO2NH2

SO2NH2

SO2NH2

NH2 NH2

1

2

4

3

SO2NH2

SO2NH2

CH2NH2

CH2CH2NH2

SO2NH2

SO2NH2

F 6

5

OH Cl

Br

Cl SO2NH2 NH2

10

N SO2NH2

HN

SO2NH2

(CH2)nOH

COOH

15: n = 0 16: n = 1 17: n = 2

18

N S

SO2NH2

14

SO2NH2

O H2N

N NH2

12

SO2NH2

13

H N

SO2NH2 NH2

11

H3C

N N

N

SO2NH2

CF3

Cl

9

S

8

SO2NH2

NH2

H2N

NH2

7

SO2NH2

SO2NH2

Cl

NH2

N N

S N H

S

SO2NH2

O

19

20

O O2N

S N H O HO

21

O SO2NH2 H2N

( )n S N H O 22: n = 0 23: n = 1 24: n = 1

SO2NH2

Fig. 1. Sulfonamides/sulfamates investigated as CAIs in the present work.

H3C

N N CH3CONH

N SO2NH2

S

CH3CON

N SO2NH2

S

AAZ

EtO

S

MZA

SO2NH2

Cl

N

EZA

NHEt

NHEt SO2NH2

SO2NH2

Me

Cl

S

S O

DCP

MeO(CH2)3

O DZA

S N H O

S

N

SO2NH2 S

S

O

O BRZ

N N

O

SO2NH2

O NH2 O S O O

O

SO2NH2

SO2NH2 O

O

N

O BZA

O

TPM ZNS

OMe O N H

H N N

Cl

O O S 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

S

SO 2NH2 O

F3C CLX

SLT

HCT

Fig. 1 (continued). Sulfonamides/sulfamates investigated as CAIs in the present work.

Sulfonamide inhibition studies of the η-class carbonic anhydrase from the malaria pathogen Plasmodium falciparum

Daniela Vullo, Sonia Del Prete, Gillian M. Fisher, Katherine T. Andrews, Sally-Ann Poulsen, Clemente Capasso,* and Claudiu T. Supuran* N SO2NH2 EtO

S

N S O

KI (PfCA) = 131 nM

SO2NH2

O

KI (PfCA) = 132 nM