Seppo Parkkila

Design, synthesis, and evaluation of hydroxamic acid derivatives as promising agents for the management of Chagas disease.

Today, there are approximately 8 million cases of Chagas disease in the southern cone of South America alone, and about 100 million people are living with the risk of becoming infected. The present pharmacotherapy is sometimes ineffective and has serious side effects. Here, we report a series of 4,5-dihydroisoxazoles incorporating hydroxamate moieties, which act as effective inhibitors of the carbonic anhydrase (CA) from Trypanosoma cruzi (TcCA). One compound (5g) was evaluated in detail and shows promising features as an antitrypanosomal agent. Excellent values for the inhibition of growth for all three developmental forms of the parasite were observed at low concentrations of 5g (IC50 values from 7.0 to <1 μM). The compound has a selectivity index (SI) of 6.7 and no cytotoxicity to macrophage cells. Preliminary in vivo data showed that 5g reduces bloodstream parasites and that all treated mice survived; it was also more effective than the standard drug benznidazole.

Carbonic Anhydrase Related Proteins: Molecular Biology and Evolution

The catalytically inactive isoforms of α-carbonic anhydrases are known as carbonic anhydrase related proteins (CARPs). The CARPs occur independently or as domains of other proteins in animals (both vertebrates and invertebrates) and viruses. The catalytic inactivity of CARPs is due to the lack of histidine residues required for the coordination of the zinc atom. The phylogenetic analysis shows that these proteins are highly conserved across the species. The three CARPs in vertebrates are known as CARP VIII, X and XI. CARPs orthologous to CARP VIII are found in deuterostome invertebrates, whereas protostomes only possess orthologs of CARP X. The CA-like domains of receptor-type protein tyrosine phosphatases (PTPR) are found only in PTPRG and PTPRZ. Most of these CARPs are predominantly expressed in central nervous system. Among the three vertebrate CA isoforms, CARP VIII is functionally associated with motor coordination in human, mouse and zebrafish and certain types of cancers in humans. Vertebrate expression studies show that CARP X is exclusively expressed in the brain. CARP XI is only found in tetrapods and is highly expressed in the central nervous system (CNS) of humans and mice and is also associated with several cancers. CARP VIII, PTPRZ and PTPRG have been shown to coordinate the function of other proteins by protein-protein interaction, and viral CARPs participate in attachment to host cells, but the precise biological function of CARPs X and XI is still unknown. The findings so far suggest many novel functions for the CARP subfamily, most likely related to binding to other proteins.

The structural comparison between membrane-associated human carbonic anhydrases provides insights into drug design of selective inhibitors.

Carbonic anhydrase isoform XIV (CA XIV) is the last member of the human (h) CA family discovered so far, being localized in brain, kidneys, colon, small intestine, urinary bladder, liver, and spinal cord. It has recently been described as a possible drug target for treatment of epilepsy, some retinopathies as well as some skin tumors. Human carbonic anhydrase (hCA) XIV is a membrane‐associated protein consisting of an N‐terminal extracellular domain, a putative transmembrane region, and a small cytoplasmic tail. In this article, we report the expression, purification, and the crystallographic structure of the entire extracellular domain of this enzyme. The analysis of the structure revealed the typical α‐CA fold, in which a 10‐stranded β‐sheet forms the core of the molecule, while the comparison with all the other membrane associated isoforms (hCAs IV, IX, and XII) allowed to identify the diverse oligomeric arrangement and the sequence and structural differences observed in the region 127–136 as the main factors to consider in the design of selective inhibitors for each one of the membrane associated α‐CAs.

Drosophila melanogaster: a model organism for controlling Dipteran vectors and pests

Abstract Beta-carbonic anhydrases (β-CAs) have been recently reported to be present in many protozoan and metazoan species, whereas it is absent in mammals. In this review, we introduce β-CA from Drosophila melanogaster as a model enzyme for pesticide development. These enzymes can be targeted with various enzyme inhibitors, which can have deleterious effects on pathogenic and other harmful organisms. Therefore, β-CAs represent a new potential target to fight against Dipteran vectors and pests relevant to medicine, veterinary medicine, and agriculture.

Bioinformatic analysis of beta carbonic anhydrase sequences from protozoans and metazoans

BackgroundDespite the high prevalence of parasitic infections, and their impact on global health and economy, the number of drugs available to treat them is extremely limited. As a result, the potential consequences of large-scale resistance to any existing drugs are a major concern. A number of recent investigations have focused on the effects of potential chemical inhibitors on bacterial and fungal carbonic anhydrases. Among the five classes of carbonic anhydrases (alpha, beta, gamma, delta and zeta), beta carbonic anhydrases have been reported in most species of bacteria, yeasts, algae, plants, and particular invertebrates (nematodes and insects). To date, there has been a lack of knowledge on the expression and molecular structure of beta carbonic anhydrases in metazoan (nematodes and arthropods) and protozoan species.MethodsHere, the identification of novel beta carbonic anhydrases was based on the presence of the highly-conserved amino acid sequence patterns of the active site. A phylogenetic tree was constructed based on codon-aligned DNA sequences. Subcellular localization prediction for each identified invertebrate beta carbonic anhydrase was performed using the TargetP webserver.ResultsWe verified a total of 75 beta carbonic anhydrase sequences in metazoan and protozoan species by proteome-wide searches and multiple sequence alignment. Of these, 52 were novel, and contained highly conserved amino acid residues, which are inferred to form the active site in beta carbonic anhydrases. Mitochondrial targeting peptide analysis revealed that 31 enzymes are predicted with mitochondrial localization; one was predicted to be a secretory enzyme, and the other 43 were predicted to have other undefined cellular localizations.ConclusionsThese investigations identified 75 beta carbonic anhydrases in metazoan and protozoan species, and among them there were 52 novel sequences that were not previously annotated as beta carbonic anhydrases. Our results will not only change the current information in proteomics and genomics databases, but will also suggest novel targets for drugs against parasites.

Expression Pattern of Carbonic Anhydrase IX in Medullary Thyroid Carcinoma Supports a Role for RET-Mediated Activation of the HIF Pathway

Medullary thyroid carcinoma is a relatively rare tumor with poor prognosis and therapy response. Its phenotype is determined by both genetic alterations (activating RET oncoprotein) and physiological stresses, namely hypoxia [activating hypoxia-inducible factor (HIF)]. Here, we investigated the cooperation between these two mechanisms. The idea emerged from the immunohistochemical analysis of carbonic anhydrases (CA) IX and XII expression in thyroid cancer. Although CAXII was present in all types of thyroid carcinomas, CAIX, a direct HIF target implicated in tumor progression, was associated with aggressive medullary and anaplastic carcinomas, and its expression pattern in medullary thyroid carcinomas suggested contribution of both hypoxic and oncogenic signaling. Therefore, we analyzed the CA9 promoter activity in transfected tumor cells expressing RET and/or the HIF-α subunit. We showed that overexpression of both wild-type and mutant RET can increase the CA9 promoter activity induced by HIF-1 (but not HIF-2) in hypoxia. Similar results were obtained with another HIF-1-regulated promoter derived from the lactate dehydrogenase A gene. Moreover, inhibition of the major kinase pathways, which transmit signals from RET and regulate HIF-1, abrogated their cooperative effect on the CA9 promoter. Thus, we brought the first experimental evidence for the crosstalk between RET and HIF-1 that can explain the increased expression of CAIX in medullary thyroid carcinoma and provide a rationale for therapy simultaneously targeting both pathways.

Expression Patterns and Subcellular Localization of Carbonic Anhydrases Are Developmentally Regulated during Tooth Formation

Carbonic anhydrases (CAs) play fundamental roles in several physiological events, and emerging evidence points at their involvement in an array of disorders, including cancer. The expression of CAs in the different cells of teeth is unknown, let alone their expression patterns during odontogenesis. As a first step towards understanding the role of CAs during odontogenesis, we used immunohistochemistry, histochemistry and in situ hybridization to reveal hitherto unknown dynamic distribution patterns of eight CAs in mice. The most salient findings include expression of CAII/Car2 not only in maturation-stage ameloblasts (MA) but also in the papillary layer, dental papilla mesenchyme, odontoblasts and the epithelial rests of Malassez. We uncovered that the latter form lace-like networks around incisors; hitherto these have been known to occur only in molars. All CAs studied were produced by MA, however CAIV, CAIX and CARPXI proteins were distinctly enriched in the ruffled membrane of the ruffled MA but exhibited a homogeneous distribution in smooth-ended MA. While CAIV, CAVI/Car6, CAIX, CARPXI and CAXIV were produced by all odontoblasts, CAIII distribution displayed a striking asymmetry, in that it was virtually confined to odontoblasts in the root of molars and root analog of incisors. Remarkably, from initiation until near completion of odontogenesis and in several other tissues, CAXIII localized mainly in intracellular punctae/vesicles that we show to overlap with LAMP-1- and LAMP-2-positive vesicles, suggesting that CAXIII localizes within lysosomes. We showed that expression of CAs in developing teeth is not confined to cells involved in biomineralization, pointing at their participation in other biological events. Finally, we uncovered novel sites of CA expression, including the developing brain and eye, the olfactory epithelium, melanoblasts, tongue, notochord, nucleus pulposus and sebaceous glands. Our study provides important information for future single or multiple gene targeting strategies aiming at deciphering the function of CAs during odontogenesis.

Effect of Genetic Polymorphisms in CA6 Gene on the Expression and Catalytic Activity of Human Salivary Carbonic Anhydrase VI

Carbonic anhydrase isoenzyme VI (CA VI) plays an important role in the homeostasis of oral tissues participating in the processes of taste, protection of dental tissues against the loss of minerals, caries, and possibly in the formation of dental calculus in periodontal disease. This study aimed to verify the correlation between changes in the expression and activity of human salivary carbonic anhydrase VI and genetic polymorphisms in its gene (CA6). The study population consisted of 182 healthy volunteers (female and male, aged 18-22). Samples of total saliva were assayed for CA VI concentrations using a specific time-resolved immunofluorometric assay. CA VI catalytic activity was detected by a modified protocol of Kotwica et al. [J Physiol Pharmacol 2006;57(suppl 8):107-123], adapted to CA VI in saliva. Samples of genomic DNA were genotyped for polymorphisms rs2274327 (C/T), rs2274328 (A/C) and rs2274333 (A/G) by TaqMan® SNP Genotyping Assays. The concentration and catalytic activity of the salivary CA VI obtained for the different genotypes were analyzed using the Kruskal-Wallis nonparametric test and the Dunn test. The results showed that individuals with TT genotype (rs2274327) had significantly lower CA VI concentrations than the individuals with genotypes CT or CC (p < 0.05). There was also an association between polymorphism rs2274333 and salivary CA VI concentrations. There were no associations between the three polymorphisms analyzed and variations in CA VI activity. Our results suggest that polymorphisms in the CA6 gene are associated with the concentrations of secreted CA VI.

Expression of cancer‐related carbonic anhydrases IX and XII in normal skin and skin neoplasms

Purpose of the study was to evaluate the presence of hypoxia‐inducible, tumour‐associated carbonic anhydrases IX and XII in normal skin and a series of cutaneous tumours. Human tumour samples were taken during surgical operations performed on 245 patients and were immunohistochemically stained. A histological score value was calculated for statistical analyses which were performed using SPSS for Windows, versions 17.0 and 20.0. In normal skin, the highest expression of CA IX was detected in hair follicles, sebaceous glands, and basal parts of epidermis. CA XII was detected in all epithelial components of skin. Both CA IX and CA XII expression levels were significantly different in epidermal, appendigeal, and melanocytic tumour categories. Both CA IX and XII showed the most intense immunostaining in epidermal tumours, whereas virtually all melanocytic tumours were devoid of CA IX and XII immunostaining. In premalignant lesions, CA IX expression significantly increased when the tumours progressed to more severe dysplasia forms. Both CA IX and XII are highly expressed in different epithelial components of skin. They are also highly expressed in epidermal tumours, in which CA IX expression levels also correlate with the dysplasia grade. Interestingly, both isozymes are absent in melanocytic tumours.

Role of carbonic anhydrases in skin wound healing

Skin wound closure occurs when keratinocytes migrate from the edge of the wound and re-epithelialize the epidermis. Their migration takes place primarily before any vascularization is established, that is, under hypoxia, but relatively little is known regarding the factors that stimulate this migration. Hypoxia and an acidic environment are well-established stimuli for cancer cell migration. The carbonic anhydrases (CAs) contribute to tumor cell migration by generating an acidic environment through the conversion of carbon dioxide to bicarbonate and a proton. On this basis, we explored the possible role of CAs in tissue regeneration using mouse skin wound models. We show that the expression of mRNAs encoding CA isoforms IV and IX are increased (~25 × and 4 ×, respectively) during the wound hypoxic period (days 2–5) and that cells expressing CAs form a band-like structure beneath the migrating epidermis. RNA-Seq analysis suggested that the CA IV-specific signal in the wound is mainly derived from neutrophils. Due to the high level of induction of CA IV in the wound, we treated skin wounds locally with recombinant human CA IV enzyme. Recombinant CA IV significantly accelerated wound re-epithelialization. Thus, CA IV could contribute to wound healing by providing an acidic environment in which the migrating epidermis and neutrophils can survive and may offer novel opportunities to accelerate wound healing under compromised conditions.