Enzymes最新文献

The biochemical machinery of all viruses comprises enzymes able to cleave polyproteins formed after the transcription of the viral genetic material, which belong to the protease class. Viral proteases known so far belong to the aspartic, serine and cysteine protease classes, with no viral metalloprotease described to date. The tridimensional structure, biochemical properties and susceptibility to be inhibited by various classes of compounds for many such enzymes have been investigated in detail in the last decades. Many antiviral drugs target viral proteases which produce diseases in mammals, but such enzymes are also present in viruses which attack plants or bacteria, and potential applications for such enzymes or their inhibition started to be considered in recent years. The aspartic protease encoded in the HIV genome, the serine proteases found in various HCV serotypes and more recently the two cystein proteases from coronaviruses, including SARS CoV 2, are targeted by clinically used drugs belonging to the protease inhibitors, which effectively interrupt the life cycle of the virus, alone or in combination therapies with other antivirals and showed a relevant clinical success. Many other less investigated viruses encode for proteases belonging to the three classes mentioned above and they started to be investigated for obtaining novel antivirals for the management of Dengue, Zika, West Nile and other flaviviruses infections but also Chikungunya, Ebola, Marbug and various other filoviruses, for which few therapeutic options are available to date.

Hepatitis C virus (HCV) is a bloodborne, hepatotropic RNA virus and a serious global health burden, infecting over 50 million people worldwide, with the majority residing in low- and middle-income countries (LMICs), that significantly contributes to chronic liver diseases like cirrhosis and hepatocellular carcinoma. While no vaccines are available for HCV yet, over the years the success of direct-acting antivirals (DAAs) has been contributing with improved cure rates among treatment-naïve and treatment-experienced patients. However, many challenges remain due to undiagnosed infections and limited access to DAAs in LMICs. Thus, understanding of the molecular biology of HCV is pivotal in driving therapeutic advances, particularly the characterization of its two major proteases: the NS2/3 protease, a cysteine protease responsible for the first cleavage event of the polyprotein; and the NS3/4A protease, a serine protease that cleaves the remainder of the HCV genome. These proteases have been extensively studied as drug targets, although there is still much to learn. The NS3/4A protease has been a validated target for the development of DAAs, with several FDA-approved drugs in recent years. Yet, many challenges remain, as the genetic diversity of HCV has been leading to the emergence of drug-resistant strains that require the administration of the costly pan-genotypic DAAs. In this chapter we explore the structure and functions of NS2/3 and NS3/4A in the viral life cycle, how DAAs engage such targets and which mutations drive resistance. We conclude by discussing future and even less explored approaches in hopes of contributing to the current HCV drug development scenario.

The biochemical machinery of most viruses comprises proteases which are crucial for their life cycle. In the last decades, proteases from pathogenic viruses started to be considered as potential drug targets, and this led to the development of several classes of effective antivirals used for the management of HIV, HCV and SARS CoV 2 infections. More than 25 clinically used protease inhibitors (PIs) are now available for the management of these three infections, but many other viruses encode for proteases which started to be considered only recently as potential drug targets. They include enterovirises, filoviruses such as Zika, Dengue and West Nile viruses, Chikungunya and other togaviruses, Ebola, Marbug and many other hemorrhagic viruses. The proteases of many such pathogens have been cloned, characterized and in some cases also crystallized in complex with inhibitors, but no compounds progressed yet to clinical trials. There are several relevant challenges in designing PIs as novel antivirals, such as: (i) the drug design strategies of peptidomimetic inhibitors, which are many times complex and expensive; (ii) the difficulties in identifying non-peptidomimetic PIs; (iii) the selectivity for the target versus host proteases of the identified PIs; (iv) their metabolism, absorption and in vivo antiviral activity, and, most importantly, (v) the emergence of drug/multidrug resistance due to the high mutation rates of many viruses. Many of these challenges started to be approached by innovative strategies which will be duscussed in the chapter.

Carbonic anhydrases (CAs) are widely distributed in the fungal kingdom and play crucial roles for their growth, development, virulence, and survival. Known fungal CAs belong either to the α- or the β-classes, with the α-class encoded only in filamentous ascomycetes. Here we report the main findings relative to α-CAs characterized so far from different fungi, namely Aspergillus oryzae, Sordaria macrospora and Paracoccidioides. Structural, functional and biochemical data will be discussed underlying the necessity of more research efforts to gain a comprehensive understanding of fungal α-CAs.

The SARS-CoV-2 main protease (M^pro) plays a pivotal role in the viral life cycle by cleaving polyproteins pp1a and pp1ab into functional non-structural proteins (NSPs), including components essential for RNA replication, such as nsp7, nsp8, and RNA-dependent RNA polymerase. The high sequence conservation across coronaviruses and absence of closely related human proteases make M^pro an attractive target for selective antiviral interventions. Recent efforts in drug discovery have led to the development of a wide spectrum of M^pro inhibitors, including covalent peptidomimetics (e.g., nirmatrelvir) and non-covalent small molecules with enhanced pharmacological profiles, such as ensitrelvir. Structure-based drug design, fragment-based drug discovery (FBDD), high-throughput screening (HTS), and in silico approaches have contributed to identification of novel scaffolds and optimization of binding interactions within the catalytic pocket. Non-covalent inhibitors offer reversible binding mechanisms that reduce off-target effects and are particularly promising for clinical translation. However, challenges such as the limited oral bioavailability of peptidomimetic compounds, metabolic instability, and emerging resistance highlight the need for further optimization. Ongoing research is exploring prodrug strategies, advanced delivery systems, and combinatorial regimens that integrate M^pro inhibitors with other antivirals to achieve synergistic effects and suppress resistance. This chapter provides a comprehensive overview of the current landscape of M^pro-targeted therapeutics and emphasizes their potential role in future pandemic preparedness.

Carbonic anhydrases (CAs) are essential metalloenzymes that catalyse the reversible conversion of CO₂ to bicarbonate, playing a crucial role in pH regulation, CO₂ sensing, and metabolic homeostasis. In Malassezia species, β-class CAs have emerged as promising drug targets for antifungal and dermatological applications, particularly in conditions such as dandruff and seborrheic dermatitis. Among the studied Malassezia species, the carbonic anhydrases from M. globosa (MgCA), M. restricta (MreCA) and M. pachydermatis (MpaCA) have been extensively characterized, demonstrating significant functional differences in both inhibition and activation mechanisms. This chapter explores the inhibition of Malassezia CAs using diverse classes of inhibitors, including sulfonamides, boronic acids, phenols, dithiocarbamates, and benzoxaboroles. Many of these compounds exhibit selective inhibition of fungal CAs over human isoforms, underscoring their potential as novel antifungal agents. Additionally, activation studies have revealed that both MgCA and MreCA can be modulated by biogenic amines and amino acids, with MreCA displaying markedly higher sensitivity, particularly to catecholamines like L-adrenaline, suggesting a potential link between stress responses and fungal virulence. The differential inhibition and activation profiles of Malassezia β-CAs provide valuable insights into fungal physiology, enzyme regulation, and potential therapeutic interventions. These findings establish a strong foundation for the rational design of selective inhibitors and activators that could serve as next-generation antifungal agents.

The multipurpose enzyme papain-like protease (PLpro) is crucial for both immune evasion and viral multiplication. The replication-transcription complex is formed when PLpro, encoded by nonstructural protein 3 (nsp3), cleaves the viral polyprotein to release nsp1 through nsp4. Furthermore, by eliminating ubiquitin and ISG15 from key immune signaling proteins, such as IRF3, STING, and MDA5, PLpro impairs host antiviral defenses by reducing type I interferon responses. The catalytic triad and several functional domains, including the flexible BL2 loop that regulates access to the viral polyprotein substrate and inhibitor, as well as the SUb1 and SUb2 binding sites for ISG15/Ub recognition, are structural features of PLpro. Due to these features, PLpro is a desirable target for both allosteric and active-site inhibition. Numerous prospective inhibitors have been identified through drug repurposing and natural product screening, supported by structural and computational analyses that highlight key interaction sites. The biological significance, structural intricacy, and therapeutic potential of PLpro as a dual-action antiviral target, which can inhibit viral replication and restore host immune function, are highlighted in this chapter.

In the last phase of their life cycle, bacteriophages form lytic enzymes known as bacteriophage endolysins that destroy the bacterial cell wall and liberate new virions. Endolysins have emerged as high-scope antimicrobial agents, especially against Gram-positive infections, due to their specificity, rapid action, and ability to target essential cell wall components. Because bacterial resistance to endolysins continues to be rare, their unique mode of action places them at a strategic advantage over traditional antibiotics. They can now be employed to design synthetic or chimeric endolysins with enhanced activity and broadened host range, even against Gram-negative bacteria, due to advancements made in molecular biology and protein engineering. This chapter gives a thorough analysis of the structure of bacteriophage endolysins, mode of action, classification, therapeutic applications, and challenges. Their potential in clinical medicine, agriculture, food safety, and biotechnology are also discussed, focusing on how they can serve as a viable solution to the global antibiotic resistance issue.

An overview of carbonic anhydrases (CAs) in fungi and protozoa is provided, emphasizing their evolutionary significance, functional diversity, and implications for human health. CAs are metalloenzymes that catalyze the reversible hydration of carbon dioxide, playing crucial roles in cellular homeostasis, pH regulation, and metabolic adaptation. In fungi, α- and β-class CAs are predominant, facilitating growth and virulence, particularly in pathogenic species such as Candida spp., Cryptococcus neoformans and many others. Protozoa exhibit a broader range of CA classes, including the recently identified η-class in Plasmodium falciparum, which is vital for the survival of the parasite and presents a significant potential as a drug target. The evolutionary trajectories of CAs reflect adaptations to diverse ecological niches, with gene duplication leading to functional diversification. Understanding the biochemical properties and regulatory mechanisms of CAs in these organisms can lead to innovative therapeutic strategies against fungal and protozoan infections, highlighting their potential as drug and diagnostic targets.

The HIV protease (HPR) is a virus-specific aspartic protease responsible for processing the polyproteins of gag and gag-pol during virion maturation and for the proliferation of HIV. The activity of HPR is essential for virus infectivity, thus it is an important target for the development of anti-HIV drugs. HPR is only one major viral protease, since there are other proteases, which are specific to HCV or SARS-CoV-2 and are therapeutic targets as well. HPR inhibitors in combination with other classes of anti-HIV drugs are one of the main components of an effective anti-HIV therapy. Nevertheless, upon several circumstances, HIV can develop a discrete pattern of resistance towards one or several HPR inhibitors through the phenomenon of cross-resistance. The aim of our work is to illustrate various features of HPR: its structure, the various mechanisms which lead to its inhibition, the HPR inhibitors which are used in the clinical arena, and the pathways involved in drug resistance, plus the mechanisms to overcome it.