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Amoebiasis, caused by Entamoeba histolytica, remains a major public health issue, particularly in developing countries with poor sanitation. It is also a significant challenge among those who travel to endemic areas, causing, in many cases, so-called traveler diarrhea. Approximately 10 percent of the global population is estimated to be affected by this parasitic infection. The primary route of transmission is the consumption of food or water contaminated with E. histolytica cysts. While most infected individuals may remain asymptomatic, some develop severe complications, including hemorrhagic colitis, liver abscesses, and, in extreme cases, colonic perforation. It has been estimated that amoebiasis is responsible for nearly 100,000 deaths annually. Standard treatment for amoebic colitis involves a combination of luminal agents (such as paromomycin, diloxanide furoate, and diiodohydroxyquin) and tissue amoebicides (including metronidazole and tinidazole). Although these treatments are effective, new therapeutic options to improve patient outcomes are needed. One promising avenue for drug discovery is the β-carbonic anhydrase enzyme (EhiCA) of E. histolytica, which has emerged as a potential target for novel antiamoebic therapies. EhiCA was recently produced as a recombinant protein and has been used in kinetic and inhibition studies with various sulfonamides and anions, with promising results.

Viral proteases are essential enzymes that orchestrate the maturation of viral polyproteins, a critical step in infectious viral particles. Understanding their structure and function is paramount for developing effective antiviral therapies. This chapter provides an overview of the central role of viral proteases in the viral lifecycle, their classification, and the wealth of structural information available through resources like the Protein Data Bank (PDB) and the MEROPS database. We discuss the catalytic mechanisms of key protease classes and highlight the remarkable structural conservation observed across different viruses and even with host proteases, providing insights into viral evolution. Furthermore, the chapter offers practical guidance on utilizing publicly available structural biology resources to facilitate research and drug discovery efforts targeting these crucial viral enzymes, empowering researchers to independently explore this evolving field.

Carbonic anhydrases (CAs, EC 4.2.1.1) were characterized in several fungi (Cryptococcus neoformans, Candida albicans and C. glabrata, Saccharomyces cerevisiae, Malassezia globosa, M. restricta and M. pachydermatis, Sordaria macrospora, Aspergillus fumigatus and A. oryzae) and protozoans (Trypanosoma cruzi, Leishmania donovani chagasi, Plasmodium falciparum, Entamoeba histolytica, Trichomonas vaginalis, Toxoplasma gondii) being also shown that they are present in Acanthamoeba castellanii. These enzymes belong to various genetic families (α- and β-CAs for fungi, α-, β-, γ- and η-classes for protozoans), showed significant CO₂ hydrase activity and a vast number of inhibitors were detected belonging to the inorganic anions, sulfonamides, phenols, mono-/dithiocarbamates, boronic acids, benzoxaboroles, or coumarins. However, few of them showed anti-infective properties in vivo or ex vivo, due to the limited number of such studies. Promising results were however obtained with sulfonamides showing antimalarial, anti-Malassezia spp., anti-T. cruzi and anti-leishmanial action against various strains of these pathogens, sometimes resistant to clinically used drugs. The main challenges for obtaining effective antifungals/antiprotozoan agents based on CA inhibitors are: (i) the complex life cycles of most of these pathogens, which frequently have different stages, hosts and diverse gene expression and metabolic patterns; (ii) lack of detailed structural data for many such enzymes; (iii) lack of focused drug design campaigns for the specific enzymes found in these pathogens, and (iv) lack of simple, inexpensive in vivo models for their testing. Future work in the field that should address these limitations might lead to relevant developments for obtaining novel anti-infectives.

Trichomoniasis is the most common sexually transmitted infection. It is caused by the parasite Trichomonas vaginalis. Nitroimidazoles, particularly metronidazole and tinidazole, have been the main treatment options for decades. They still remain the standard treatment, and resistance to them is relatively rare. However, cases of resistance do occur, and the side effects can be significant. This highlights the urgent need for new drugs with different mechanisms of action. Promisingly, several innovative leads have emerged. Interesting drug targets in T. vaginalis include two β-carbonic anhydrases, which have been recently described. These enzymes have been characterized in terms of their structural and kinetic properties, and potential inhibitors have been identified. This new knowledge on β-carbonic anhydrases offers hope for the development of novel antitrichomonal agents to effectively combat this parasitic disease in the future.

Carbonic anhydrases are ubiquitous metalloenzymes which catalyze the CO₂ hydration to bicarbonate and proton. β-Carbonic Anhydrases from fungi, such as Saccharomyces cerevisiae, Candida spp. and Cryptococcus neoformans, have been widely investigated as potential targets for antifungal therapies. In this chapter, we provide a comprehensive overview on their properties highlighting their role as CO₂-sensing enzymes. We survey functional, biochemical, structural, and kinetic features, summarize inhibition and activation studies, and review in vitro experiments. Taken together, these data underscore fungal β‑carbonic anhydrases as promising potential targets for the development of new antifungal strategies.

Malaria parasites belonging to the genus Plasmodium encode for a carbonic anhydrase (CA, EC 4.2.1.1) originally considered to belong to the α-class, which has been investigated starting with 2004 as a potential antimalarial target, considering the observation that CA levels in red blood cells infected with these parasites are much higher compared to those of uninfected cells. In plasmodia, CA is involved in metabolic pathways leading to the biosynthesis of pyrimidines, which are scarcely present in the blood of infected hosts, making this enzyme crucial for the life cycle of the parasite in many intraerythrocytic stages of its development. It has been then shown in 2014 that P. falciparum CA (PfCA) belongs in fact to a new CA genetic class, the η-CA, characterized by a particular zinc ion coordination within the active site, with two histidine and a glutamine as protein ligands. A short, truncated and longer PfCA forms have been cloned and characterized in detail, being shown that they act as efficient catalysts for the hydration of CO₂ to bicarbonate and protons, but neither of them were crystallized for the moment, and their 3D structure is not known. PfCA inhibition with anions, sulfonamides, phenols and coumarins has been investigated too, with many low nanomolar in vitro inhibitors being detected. Only for acetazolamide and an ureido-substituted benzenesulfonamide it has been demonstrated a potent growth inhibition of the pathogen in P. falciparum infected red blood cells. Although these results are encouraging but rather preliminary, η-CAs from malaria-producing protozoans and presumably other organisms encoding them, may be considered as innovative drug targets for obtaining anti-infectives with new mechanisms of action but these enzymes should be investigated in more details in order to better understand their structure and physiological/pathological roles.

Viral proteases are critical enzymes that play essential roles in the replication of viruses such as Human Immunodeficiency, Hepatitis C, SARS-CoV-2, Zika, Dengue, West Nile, Yellow Fever, Japanese and Saint Louis Encephalitis, Tick-Born Encephalitis, Chikungunya, and others. Designing potent inhibitors against these proteases has been a major therapeutic strategy to control and treat these viral infections. Computational approaches, including structure-based drug design, ligand-based drug design, machine learning and artificial intelligence-based techniques, have significantly accelerated the discovery and optimization of viral protease inhibitors. This chapter provides an in-depth review of the computational methodologies employed in the development of inhibitors for these major viral targets, highlighting case studies for each virus, discussing strategies to overcome resistance, and exploring future directions in antiviral drug discovery.

Carbonic anhydrases (CAs) play an essential role in the physiology and survival of protozoan parasites. This study explores the biological functions, molecular features, and therapeutic potential of protozoan CAs, focusing on the α, β, and η classes. Emphasis is placed on the structural and functional divergences between protozoan and mammalian CAs, underscoring the opportunities for selective drug targeting. Key protozoan pathogens, including Toxoplasma gondii, Trypanosoma cruzi, Leishmania spp., Trichomonas vaginalis, Entamoeba histolytica and Plasmodium falciparum, are examined with respect to their CA classes, which are evaluated for their roles in parasite metabolism and as candidates for therapeutic intervention. The potential of CA inhibitors as novel antiparasitic agents was critically assessed. By integrating established findings with emerging data, this analysis offers a comprehensive framework for the strategic exploitation of protozoan CAs for the development of next generation antiparasitic therapies.

Sordaria macrospora, a coprophylous fungus used for the last three decades as a model organism for studying fruiting body development of fungi, encodes for four carbonic anhydrases (CAs, EC 4.2.1.1), CAS1-CAS4. CAS1-CAS3 are β-CAs and were investigated in detail in the last years, whereas CAS4, an α-class enzyme, was less investigated. All of them are crucial for the fungus, as the mutant lacking the genes encoding for these four enzymes showed a drastically reduced vegetative growth rate compared to the wild type organism. CAS4 is a secreted protein, CAS2 is mitochondrial, whereas CAS1 and CAS3 are cytosolic enzymes. The catalytic activity of CAS1-CAS3 for the CO₂ hydration reaction showed that all of them possess a significant activity, with CAS3 being the most effective catalyst. The X-ray crystal structures of CAS1 and CAS2 were also obtained, showing that the two enzymes are tetramers (dimers of dimers) with an open active site in the case of CAS1 and a closed one for CAS2, similar to other plant/fungal/bacterial β-CAs studied so far. Detailed anion and sulfonamide inhibition studies were reported for all three β-Cas, which led to the identification of several effective inhibitors. Potential biotechnological applications of these enzymes for carbon (CO₂) capture are also discussed.

West Nile virus (WNV), Zika virus (ZIKV), and Dengue virus (DENV) are vector-borne diseases endemic in tropical and subtropical countries around the world. Their incidence has been growing in recent years and they are becoming increasingly relevant even in non-endemic areas, representing a significant public health problem worldwide. Globalization and climate change have led to drastic changes and radical transformations in the development of an ecosystem capable of sustaining the life cycle of viruses even in urban environments, where transmission occurs within an entirely susceptible population, leading to the rapid spread of infection, which can take on an explosive epidemic pattern. Increasing urbanization, population growth, and the continuous evolution of pathogens are additional factors associated with the increasing spread of vector-borne infectious diseases. Our focus is on these three vector-borne diseases, the diagnosis of which is often challenging because geographical and clinical overlap and serological cross reactions makes differential diagnosis very difficult.