Identification and inhibition of carbonic anhydrases from nematodes

Abstract

Carbonic anhydrases (CAs) are metalloenzymes, and classified into the evolutionarily distinct α, β, γ, δ, ζ, and η classes. α-CAs are present in many living organisms. β- and γ-CAs are expressed in most prokaryotes and eukaryotes, except for vertebrates. δ- and ζ-CAs are present in phytoplanktons, and η-CAs have been found in Plasmodium spp. Since the identification of α- and β-CAs in Caenorhabditis elegans, the nematode CAs have been considered as an emerging target in research focused on antiparasitic CA inhibitors. Despite the presence of α-CAs in both helminths and vertebrates, structural studies have revealed different kinetic and inhibition results. Moreover, lack of β-CAs in vertebrates makes this enzyme as an attractive target for inhibitory studies against helminthic infection. Some CA inhibitors, such as sulfonamides, have been evaluated against nematode CAs. This review article aims to present comprehensive information about the nematode CAs and their inhibitors as potential anthelminthic drugs.

Keywords:

• Acetazolamide
• carbonic anhydrase
• carbonic anhydrase inhibitors
• nematode
• sulfonamide

Introduction

Carbonic anhydrases (CAs, EC 4.2.1.1) are classified into six evolutionarily distinct classes: α, β, γ, δ, ζ, and η1,2. The active sites of these enzymes contain most commonly a zinc ion (Zn²⁺), which plays a crucial role in the catalytic activity of the metalloenzymes3. ζ and γ-CAs contain cadmium (ζ), iron (γ), or cobalt (γ) as cofactors3,4 instead of Zn²⁺. CAs catalyze the reversible hydration of carbon dioxide (CO₂) to bicarbonate ions (HCO₃⁻) and protons (H⁺). α-CAs are present in many prokaryotic and eukaryotic organisms, such as vertebrates, invertebrates, and plants. In total, 13 enzymatically active α-CAs have been discovered in mammals, including: CA I, CA II, CA III, CA IV, CA VA, CA VB, CA VI, CA VII, CA IX, CA XII, CA XIII, CA XIV, and CA XV5. β-CAs are expressed in many archaea, prokaryotes, and eukaryotes (fungi, algae, plants, protozoans, arthropods, and nematodes)5–10. γ-CAs are found in archaea, plants, and some bacteria. δ- and ζ-CAs are present in many species of marine phytoplankton5. The newly discovered η-CAs are the only genus specific CAs, present in Plasmodium spp. (the causative protozoan of malaria), and contain Zn²⁺in their catalytic active site2. CAs catalyze several important metabolic and biochemical functions, such as pH homeostasis11, electrolyte transfer12, calcification13, gluconeogenesis14, lipogenesis15, and ureagenesis16. β-CA is a critical metalloenzyme that catalyzes many biological pathways in both prokaryotes and eukaryotes including protozoans, insects, and nematodes. β-CA produces bicarbonate for carboxylases in Corynebacterium glutamicum17, urease in H. pylori18, and cyanase to detoxify cyanate in Escherichia coli19 and Ascaris lumbricoides20. Crystal structures of β-CAs demonstrated that two cysteines and one histidine are conserved and are ligated to a zinc ion in the catalytic active site of the enzyme21. A study from 2010 introduced two metazoan β-CAs that were encoded by two different β-CAs genes (y116a8c(0).28 and bca-1) in Caenorhabditis elegans6,22. In another study of 2010, a β-CA gene (DmBCA) was detected from Drosophila melanogaster6.

α-CAs are generally present in all metazoan species (vertebrates and invertebrates), whereas β-CAs are found only in invertebrate metazoans6 (arthropods and nematodes). Nematodes are categorized into several phyla: Annelida (segmented worms), Chaetognatha (arrow worms), Gnathostomulid (jaw worms), Hemichordata (acorn/tongue worms), Nematoda (roundworms), Nematomorpha (horsehair worms), Nemertea (ribbon worms), Onychophora (velvet worms), Phoronida (horseshoe worms), Platyhelminthes (flatworms), Priapulida (phallus worms), and Sipuncula (peanut worms). Most of α- and β-CA-containing nematodes (both pathogenic and nonpathogenic) have been classified into Nematoda and Platyhelminthes phyla, respectively23. The details of α- and β-CA expressing nematodes, platyhelminthes, annelids, and hemichordates are shown in Table 1. Among different phyla, some groups, such as Nematoda and Platyhelminthes, are very important as zoonotic helminths in both human and veterinary medicine. Some phyla, such as Hemichordata and Annelida, are generally considered as nonpathogenic nematodes. However, CAs have also been discovered in a few annelids, namely in Hirudo medicinalis24, Riftia pachyptila25, and Osedax bone worms26.

Table 1. α- and β-CA expressing nematoda, platyhelminthes, annelida, and hemichordate.

Name	Phyla	α-CA IDs from UniProt	Subcellular location*	β-CA IDs from UniProt	Subcellular location*
Ancylostoma caninum	Nematoda	–	S	ANCCAN_03834**	–
Ancylostoma ceylanicum	Nematoda	A0A016S6V2	S	–	–
Ancylostoma duodenale	Nematoda	A0A0C2HC60	–	–	–
Angiostrongylus cantonensis	Nematoda	A0A0K0DQ44	S	–	–
Anisakis simplex	Nematoda	A0A0M3KDS4	–	–	–
Ascaris lumbricoides (A. suum)	Nematoda	F1L837	S	F1LE18	–
Brugia malayi	Nematoda	A0A0K0JIY2	S	–	–
Brugia pahangi	Nematoda	A0A0N4TVP9	–	–	–
Caenorhabditis brenneri	Nematoda	B3GEK5	M	G0MSW4G0MRG1	MM
Caenorhabditis briggsae	Nematoda	A8XNL8	–	G0MSW4A8WN21	–M
Caenorhabditis elegans	Nematoda	Q27504	–	Q22460Q2YS41D3NQA9	–––
Caenorhabditis japonica	Nematoda	H2W178	S	–	–
Caenorhabditis remanei	Nematoda	E3LD70	S	E3LDN3E3MK96	–M
Clonorchis sinensis	Platyhelminthes	H2KQV0	S	–	–
Dictyocaulus viviparus	Nematoda	A0A0D8XQR7	–	A0A0D8XFQ1	–
Dracunculus medinensis	Nematoda	A0A0N4US17	–	A0A0N4UN75	–
Echinococcus granulosus	Platyhelminthes	W6UBF1	–	–	–
Enterobius vermicularis	Nematoda	A0A0N4V7Z0	–	A0A0N4V5S3	M
Haemonchus contortus	Nematoda	U6PI84	S	U6PDI1	M
Haemonchus placei	Nematoda	A0A0N4X8K2	S	–	–
Helobdella robusta	Annelida	T1FNV0	S	–	–
Hymenolepis microstoma	Platyhelminthes	A0A068X7B2	S	–	–
Loa loa	Nematoda	E1FQZ7	S	–	–
Necator americanus	Nematoda	W2TSS6	–	W2SJ13	–
Nippostrongylus brasiliensis	Nematoda	A0A0N4YJY2	S	A0A0N4XIZ5	M
Oesophagostomum dentatum	Nematoda	A0A0B1SZR2	–	–	–
Onchocerca volvulus	Nematoda	A0A044SJH1	S	–	–
Opisthorchis viverrini	Platyhelminthes	A0A074ZVU6	–	–	–
Parastrongyloides trichosuri	Nematoda	A0A0N4ZSS2	–	A0A0N4ZFD7A0A0N5A3V6	––
Pristionchus pacificus	Nematoda	H3FHA2	S	–	–
Saccoglossus kowalevskii	Hemichordata	–	–	NP_001171747.1***	M
Schistosoma haematobium	Platyhelminthes	A0A095AIF7	–	–	–
Schistosoma japonicum	Platyhelminthes	Q5DFH8	–	Sjp_0056790**	–
Schistosoma mansoni	Platyhelminthes	G4VP62	–	G4V6B2	–
Strongyloides papillosus	Nematoda	A0A0N5C6N3	–	A0A0N5B1Z3	–
Strongyloides ratti	Nematoda	A0A090KUC2	S	A0A090LV46	–
Strongyloides stercoralis	Nematoda	A0A0K0DY00	S	A0A0K0E635	–
Strongyloides venezuelensis	Nematoda	A0A0K0FAM6	S	A0A0K0EVN4	–
Syphacia muris	Nematoda	A0A0N5A923	–	A0A0N5AQS5	M
Thelazia callipaeda	Nematoda	A0A0N5D8E0	–	–	–
Toxocara canis	Nematoda	A0A0B2V882	S	A0A0B2UWQ8	–
Trichinella spiralis	Nematoda	E5SX27	S	E5SH53	M
Trichuris muris	Nematoda	A0A0N5DXC0	–	A0A0N5DWJ5	M
Trichuris suis	Nematoda	A0A085M2M2	–	A0A085MP73	–
Trichuris trichiura	Nematoda	A0A077YWY0	–	A0A077YZT0	M
Wuchereria bancrofti	Nematoda	J9EPL8	–	–	–

*Abbreviations: S (CAs containing signal peptide for the secretory pathway) and M (CAs containing mitochondrial targeting sequence).

** IDs from WormBase Parasite database (http://parasite.wormbase.org/)46.

*** IDs from NCBI database (http://www.ncbi.nlm.nih.gov/)47.

In this review article, we present recent discoveries regarding nematode α- and β-CAs. Moreover, the inhibition studies of these enzyme families are described as they represent plausible targets for designing novel anti-infective drugs.

α-CA expressing nematodes

A wide range of different α-CA proteins are expressed in nematodes. Despite only one representative α-CA protein ID shown for each species in Table 1, α-CA protein sequences can differ both within and between nematodes. Previous studies have identified α-CAs from some nematodes, including C. elegans and Ostertagia ostertagi27–29. The genome of C. elegans encodes for six α-CAs, namely CAH-1, CAH-2, CAH-3, CAH-4, CAH-5, and CAH-629, with CAH-4 present as two isoforms CAH-4a and CAH-4b. In 2011, Fasseas et al. concluded that only CAH-3, CAH-4, and CAH-5 possess the three conserved zinc-binding histidine residues which are also present in active human CAs. CAH-3 was shown to be an active enzyme, and CAH-4 was found to be active in vitro, while the other four CAs (CAH-1, CAH-2, CAH-5, and CAH-6) were presented as CA-related proteins (CARPs) without any CA activity (Figure 1). They also discovered that the silencing of cah-3 and cah-4 genes seemed to affect the lifespan of C. elegans.

Figure 1. Multiple sequence alignment (MSA) of α-CA protein sequences from nematodes. MSA of 43 and one α-CA protein sequences from nematodes and Klebsiella pneumoniae (outgroup), respectively, aligned using Clustal Omega algorithm from EMBL-EBI database (http://www.ebi.ac.uk/Tools/msa/clustalo/)45. MSA was conducted on 60 amino acids of α-CA protein sequences starting three amino acids prior to the first highly conserved histidine. The three highly conserved histidines locate in the catalytic active site of α-CAs and participate in binding with the Zn²⁺ion (shown by red arrows).

O. ostertagi (Brown Stomach Worm) is a parasitic nematode (helminth) and the causative agent of ostertagiosis in cattle. The CA gene from O. ostertagi has been named as OoCA27. The nucleotide sequence of OoCA gene is 78% and 55% identical with the cah-6 and CA3 genes of C. elegans and human, respectively. Studies have suggested that O. ostertagi CA may play a critical role in the immediate early developmental events following exsheathment initiation. Exsheathment is the first step at the beginning of O. ostertagi infection, and involves the casting of the second larval stage cuticle, which is retained by the infective third-stage larvae. Inhibition of this O. ostertagi development process may hinder the formation of the infective forms of O. ostertagi. In addition, it has been previously shown that ethoxzolamide affected the development of H. contortus via some changes in the excretory cells and esophagus during exsheathment30. The potential of parasitic α-CAs as drug targets is limited by possible or even probable effects on host α-CAs, which predispose to various adverse effects.

β-CA expressing nematodes

β-CAs from nematodes are particularly interesting, because these enzymes are not found in vertebrates (including humans). Inhibition of nematode β-CAs presents possibilities to treat or restrict many helminthic infections with minimal side effects on the hosts. Noteworthy is the fact that even though the β-CAs and other CA groups catalyze the same reaction, their protein structure is different. First, the Zn²⁺ion in the active site of β-CAs is coordinated by two cysteines and one histidine instead of three histidines (Figure 2)31. Second, β-CAs can be found in many oligomeric states, whereas α-CAs mainly occur as monomers and γ-CAs as trimers. Thus far, a variety of multimeric crystal structures, including dimeric, tetrameric, hexameric, and octameric β-CAs have been reported32–34. The monomeric components of a dimeric β-CA bind to each other usually by noncovalent interactions, and in some cases via a short polypeptide linker. The latter case is called a “pseudo-dimer”35. Tetrameric and octameric β-CAs are formed whenever dimers form dimer-of-dimers and dimer-of-dimer-of-dimers, respectively. The most frequently available quaternary structure of β-CAs is the tetrameric state. However, a dimeric β-CA seems to be the fundamental structural unit in β-CA protein structures.

Figure 2. Multiple sequence alignment (MSA) of β-CA protein sequences from nematodes. MSA of 31 and one β-CA protein sequences from nematodes and Bacillus subtilis (outgroup), respectively, aligned using Clustal Omega algorithm from EMBL-EBI database (http://www.ebi.ac.uk/Tools/msa/clustalo/)45. MSA was conducted on 115 amino acids of β-CA protein sequences starting three amino acids prior to the first highly conserved sequence (CXDXR; C: Cysteine, D: Aspartic acid, R: Arginine, D: any residue). The first (CXDXR) and second (HXXC; H: Histidine, C: Cysteine, X: any residue) highly conserved sequences locate in the catalytic active site of β-CAs and are coordinated with the Zn²⁺ion (shown by green and blue arrows, respectively).

Phylogenetic analysis of nematode α- and β-CAs

Nematode α-CAs and β-CAs were identified by BLASTP searches of the NCBI and UniProt databases using previously annotated members of both groups as query sequences, and all parameters as default. For each group, retrieved sequences were aligned using Clustal Omega and visually inspected for regions of incompleteness or poor quality. For those species containing a suspect sequence, the whole genome was downloaded and utilizing the exonerate program the remaining proposed high-quality sequences used as templates to predict gene and protein sequences. Subsequently, 14 genomes were analyzed for suspect sequences in the α-CA group and eight predictions kept, and ten genomes for the β-CA group with five predictions kept. In total, 54 α-CAs and 41 β-CAs protein sequences were aligned independently and used to perform phylogenetic analyses. The PhyML program was used to perform the phylogenetic analyses, utilizing the LG model and 1000 bootstraps; the results were visualized using FigTree and are presented as trees in Figures 3 and 4. Super-computing resources from the Center for Science and Computing of the Ministry of Finland were used to perform these analyses. Even at the amino acid level, the sequences for the nematode CAs were significantly different, which is evidenced in low bootstrap values within some subclades. Conversely, most branch points delineating subgroups of like organisms are well supported. This could imply that these species have possessed their CA sequences for a long time, and due to the fact that nontruncated versions of the sequences were used, ancillary domains accompanying the CA domain are providing a variation in function or localization of protein by species.

Figure 3. Phylogenetic analysis of nematode α-CAs. A total of 54 nematode α-CAs were aligned using Clustal Omega and used to perform a phylogenetic analysis using the LG model in the PhyML program, with 1000 bootstraps. Node values indicate bootstrap values. Any sequences which were predicted from the full genomic sequence are labeled as “predicted”.

Figure 4. Phylogenetic analysis of nematode β-CAs. A total of 41 nematode β-CAs were aligned using Clustal Omega and used to perform a phylogenetic analysis using the LG model in the PhyML program, with 1000 bootstraps. Node values indicate bootstrap values. Any sequences which were predicted from the full genomic sequence are labeled as “predicted”.

Subcellular location of nematode α- and β-CAs

The plant β-CAs have been localized to the cytoplasm of cells and thylakoid space of chloroplastic stroma36. In cyanobacteria, β-CA is localized in the carboxysome organelle, and is therefore involved in the CO₂-concentration process36,37. Subcellular localization studies have indicated that DmBCA is probably a mitochondrial enzyme6,7,38. The prediction of subcellular localization of α- and β-CAs from nematodes was performed using TargetP 1.1 Server (http://www.cbs.dtu.dk/services/TargetP/)39. The results are shown in Table 1. The prediction results revealed that many nematode α-CAs contain signal peptides which target them to the secretory pathway. The prediction results further revealed that some nematode β-CAs including those in C. brenneri (G0MSW4 and G0MRG1), C. briggsae (A8WN21), C. remanei (E3MK96), E. vermicularis (A0A0N4V5S3), H. contortus (U6PDI1), Nippostrongylus brasiliensis (A0A0N4XIZ5), S. kowalevskii (NP_001171747.1), S. muris (A0A0N5AQS5), T. spiralis (E5SH53), T. muris (A0A0N5DWJ5), and T. trichiura (A0A077YZT0) contain a mitochondrial targeting sequence. In fact, both α- and β-CAs from C. brenneri seem to contain a mitochondrial targeting sequence.

Inhibitory studies on nematode α-CAs

To evaluate the effect of a CA inhibitor, acetazolamide, on a nematode species, it was first tested against live C. elegans. The test revealed that this inhibitor could not penetrate the nematode cuticle28. Additionally, thiobendazole-5-sulfonamide (a sulfonamide derivative of thiobendazole) was tested, and it inhibited efficiently (K_i 9.5 nM) the CAH-4b of C. elegans40. Thiobendazole is widely used as an antiparasitic agent against both human and animal parasitic infections. The mechanism of action for thiobendazole is poorly understood, but it has been suggested that it interferes with the formation of cytoplasmic microtubules and cytoskeletons. Moreover, 2-(hydrazinocarbonyl)-3-substituted-phenyl-1H-indole-5-sulfonamides have been tested against C. elegans CAH-4b. Some of the tested compounds, including acetazolamide, ethoxzolamide, and a series of sulfonamide derivatives possessing various 2-, 3-, or 4-substituted phenyl groups with methyl-, halogeno-, and methoxy-functionalities, as well as the perfluorophenyl moiety, showed very significant inhibitory effects on CAH-4b with K_is between 6.0 and 13.4 nM41.

Inhibitory studies on nematode β-CAs

Inhibition data available for nematode β-CAs are still very limited. The first studies on nematode enzymes were performed on C. elegans and A. lumbricoides20,22. In 2009, Fasseas et al. characterized C. elegans β-CA for the first time22. They discovered two β-CAs in C. elegans, BCA-1, and Y116A8C.28, of which the latter was found to possess catalytic activity with k_cat and k_cat/K_m of 2.77 × 10⁴ s⁻¹ and 6383 × 10⁵M⁻¹s⁻¹, respectively. However, the sequence for BCA-1 was obviously incorrect, suggesting that both β-CAs from C. elegans might be active enzymes6. The RNAi studies of C. elegans β-CA did not reveal any visible phenotype, while silencing the β-CA gene in fruit fly Drosophila melanogaster caused complete infertility in females38. Hence, this result suggested crucial biochemical and physiological roles for β-CAs in insects, and possibly all invertebrates. β-CA has been characterized recently from A. lumbricoides, the causative agent of zoonotic ascariasis. The enzyme was identified using bioinformatic and computational biology methods. A. lumbricoides recombinant β-CA protein (AlBCA) was produced in Sf9 insect cells, and the kinetic parameters were investigated. Based on the results20, AlBCA possesses high catalytic activity with K_m 6.0 × 10⁵ s⁻¹ and k_cat/K_m 4.3 × 10⁷ M⁻¹s⁻¹. In addition, the K_i for inhibition of AlBCA by acetazolamide was 84.1 nM. Meanwhile, the kinetic and inhibition studies were also performed on the produced recombinant β-CAs from D. melanogaster (DmBCA)42, Anopheles gambiae (AgaCA)43, and Leishmania donovani9. These results are shown for comparison in Table 2.

Table 2. Enzyme activity and inhibition data of β-CAs from protozoa, nematode, and insects.

Enzyme	CA class	kcat (s^−1)	kcat/Km (M^−1 × s^−1)	Ki (acetazolamide) (nM)*
LdcCA	Β	9.35 × 10^5	5.9 × 10^7	91.7 ± 5.7
DmBCA	β	9.5 × 10^5	1.1 × 10^8	516 ± 24
AgaCA	β	7.2 × 10^5	5.6 × 10^7	27.3 ± 2.0
AlBCA	β	(6.0 ± 0.1) × 10^5	(4.3 ± 0.2) × 10^7	84.1 ± 2.9

The results include enzyme activity values and inhibition results with acetazolamide on the β-CAs from Leishmania donovani chagasi (LdcCA), Drosophila melanogaster (DmBCA), Anopheles gambiae (AgaCA), and Ascaris lumbricoides (AlBCA).

In vivo studies have not been conducted so far, but functional predictions of A. lumbricoides β-CA suggested that this enzyme may play important roles in bicarbonate dependent biosynthetic/metabolic pathways, such as gluconeogenesis and detoxification of metabolically produced cyanate by bicarbonate-dependent cyanase.

β-CAs from nematodes as vaccine candidates

The β-CA enzyme is highly distributed among infectious agents, including helminths. Therefore, it is a potential molecular target for controlling parasites and pests in all fields of human and veterinary medicine and agriculture10. Absence of β-CA in vertebrates also makes this enzyme a potential target protein for vaccines. The detection of antigenic sites of antigens and proteins is an important step in designing an effective vaccine candidate. For this purpose, 31 β-CA protein sequences from nematodes were analyzed with the European Molecular Biology Open Software Suite (EMBOSS) program Antigenic (http://emboss.bioinformatics.nl/cgi-bin/emboss/antigenic), which is based on the Kolaskar and Tongaonkar method44. The antigenic site prediction results revealed that the second highly conserved sequence (HXXC) represents an epitope with the highest score for a probable antigenic site among the nematode β-CAs (Table 3). On the other hand, a previous homology modeling study on β-CA from A. caninum defined that this epitope is located in the active site of enzyme and is mainly buried (Figure 5). In addition, most β-CAs are intracellular proteins, which make them inaccessible for immunological responses of the host. Therefore, β-CA inhibitors should be considered as a better option for developing new treatment strategies against parasitic or helminthic infections.

Figure 5. Accessibility identification of the predicted antigenic epitope of β-CA from A. caninum. The molecular surface of the homology model of A. caninum β-CA is shown as solid gray, and the second highly conserved sequence (HXXC) as the target epitope is buried from the surface of the protein. The exposed and buried residues of epitope are shown with red and green spheres and numbered. Figure adopted with author’s permission from open access¹⁰.

Table 3. Predicted antigenic sites of 31 β-CAs from nematodes.

Name	β-CA IDs from UniProt	The most antigenic epitopes
Ancylostoma caninum	ANCCAN_03834*	110 INHVIVCGHSDCKAINTLYNIHECPHTFDP 139
Ascaris lumbricoides (A. suum)	F1LE18	102 KHAIVCGHSDCKAMST 117
Caenorhabditis brenneri	G0MSW4G0MRG1	98 RHIVVCGHSDCKAINTLYGLHQCPKNF 124101 INHVIVCGHSDCKAINTLYNLHKCPKS 127
Caenorhabditis briggsae	A8XKV0A8WN21	97 IRHVVVCGHSDC 108101 INHVIVCGHSDCKAINTLYNLHKCPKS 127
Caenorhabditis elegans	Q22460Q2YS41	98 RHIVVCGHSDCKAINTLYGLHQCPKNF 124101 INHVIVCGHSDCKAINTLYNLHKCPKS 127
Caenorhabditis remanei	E3LDN3E3MK96	97 IRHVVVCGHSDCKAINTLYRLHQCPKE 123101 INHVIVCGHSDCKAINTLYNLHKCPKS 127
Dictyocaulus viviparus	A0A0D8XFQ1	101 INHVIVCGHSDCKAINTLYNIHKCPKSF 128
Dracunculus medinensis	A0A0N4UN75	82 IKHAVVCGHSDCKAMK 97
Enterobius vermicularis	A0A0N4V5S3	72 GETVLGGCGHEVSPIAEPAGLDLTVKLGKIKHV IVCGHSDCAVTLYDIY 120
Haemonchus contortus	U6PDI1	102 INHVIVCGHADCKAINTLYNLH 123
Necator americanus	W2SJ13	109 INHVIVCGHSDCKAINTLYNIHTCPQN 135
Nippostrongylus brasiliensis	A0A0N4XIZ5	98 RHIVVCGHSD 108
Parastrongyloides trichosuri	A0A0N4ZFD7A0A0N5A3V6	101 INHVIVCGHSDCKAINTLYNLH 122101 INHVIVCGHSDCKAINTLYNLH 122
Saccoglossus kowalevskii	NP_001171747.1**	98 NHVIVCGHSDC 108
Schistosoma japonicum	Sjp_0056790*	68 VTPGFLELTLIRCKITDIIICGHSDC 93
Schistosoma mansoni	G4V6B2	78 ENCVTPGFLELTLLRCRINDIIICGHSDC 106
Strongyloides papillosus	A0A0N5B1Z3	101 INHVIVCGHSDCKAINTLYNLHTGV 125
Strongyloides ratti	A0A090LV46	101 INHVIVCGHSDCKAINTLYNLHTGV 125
Strongyloides stercoralis	A0A0K0E635	101 INHVVVCGHSDCKAINTLYNLHTGV 125
Strongyloides venezuelensis	A0A0K0EVN4	101 INHVIVCGHSDCKAINTLYNLHTGV 125
Syphacia muris	A0A0N5AQS5	73 FGGCQHEVSAVAEPAGLDLTVKLGKIKHVIVCGHSDCAAMK 113
Toxocara canis	A0A0B2UWQ8	102 LKHAIVCGHSNC 113
Trichinella spiralis	E5SH53	100 KDIVVCGHSDC 110
Trichuris muris	A0A0N5DWJ5	77 SGAIAEASASLELACCVSKVPEIIVCGHSDCKAMKL 112
Trichuris suis	A0A085MP73	119 EATASLKLACCESKVPEVIVCGHSDCKAMKLLWLL 153
Trichuris trichiura	A0A077YZT0	94 TASLQLACCESKVPEVIVCGHSDCKAMKLLWS 125

*IDs from WormBase Parasatie database (http://parasite.wormbase.org/)46.

** IDs from NCBI database (http://www.ncbi.nlm.nih.gov/)47.

Conclusion

As the overall conclusion, inhibitory studies have demonstrated that acetazolamide is able to efficiently inhibit β-CA from a nematode, A. lumbricoides. Other studies have also shown that both acetazolamide and ethoxzolamide significantly inhibit an α-CA from C. elegans (CAH-4b). Even though the literature on nematode β-CAs is still rather limited, β-CAs can be considered more attractive than α-CAs as potential targets for anti-helminthic drugs. This is based on the fact that β-CAs are absent from the proteomes of vertebrates. Therefore, β-CAs could represent a helminthic-specific drug targets with minimal side effects on the infected vertebrate host.

Declaration of interest

The authors declare that they have no competing interests.

To perform the original studies, RZE received a scholarship support from the Ministry of Science, Research and Technology, and National Institute of Genetic Engineering and Biotechnology of Islamic Republic of Iran. The studies were also supported by research grants from the Academy of Finland to SP, Finnish Funding Agency for Innovation (TEKES) to SP, Finnish Cultural Foundation (Pirkanmaa Regional Fund and Maili Autio Fund) to HRB and RZE, Sigrid Juselius Foundation to SP, Jane and Aatos Erkko Foundation to SP, Tampere Tuberculosis Foundation to SP, and Competitive Research Funding of the Tampere University Hospital.