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Fungal infections represent a growing public health problem, mainly stemming from two phenomena. Firstly, certain diseases (e.g., AIDS and COVID-19) have emerged that weaken the immune system, leaving patients susceptible to opportunistic pathogens. Secondly, an increasing number of pathogenic fungi are developing multi-drug resistance. Consequently, there is a need for new antifungal drugs with novel therapeutic targets, such as type I and II DNA topoisomerase enzymes of fungal organisms. This contribution summarizes the available information in the literature on the biology, topology, structural characteristics, and genes of topoisomerase (Topo) I and II enzymes in humans, two other mammals, and 29 fungi (including Basidiomycetes and Ascomycetes). The evidence of these enzymes as alternative targets for antifungal therapy is presented, as is a broad spectrum of Topo I and II inhibitors. Research has revealed the genes responsible for encoding the Topo I and II enzymes of fungal organisms and the amino acid residues and nucleotide residues at the active sites of the enzymes that are involved in the binding mode of topoisomerase inhibitors. Such residues are highly conserved. According to molecular docking studies, antifungal Topo I and II inhibitors have good affinity for the active site of the respective enzymes. The evidence presented in the current review supports the proposal of the suitability of Topo I and II enzymes as molecular targets for new antifungal drugs, which may be used in the future in combined therapies for the treatment of infections caused by fungal organisms.

Organotin complexes were prepared through a one-pot reaction with three components by reacting thiosemicarbazide or 4-methyl-3-thiosemicarbazide or 4-phenylthiosemicarbazide, dehydroacetic acid (DHA) and dibutyl, diphenyl, dicyclohexyl, and bis[(trimethylsilyl)methyl]tin(IV) oxides; all complexes were characterized by infrared (IR), ultraviolet-visible (UV-vis), mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy. The 119Sn NMR revealed chemical shifts corresponding to a pentacoordinated environment in solution. The X-ray crystallography of the two complexes evidenced the formation of monomeric complexes with a pentacoordinated geometry around tin via three donor atoms from the ligand, the sulfur of the thiol, the nitrogen of the imine group, and the oxygen of the pyran ring. The geometries of the five-coordinated complexes 3a (Bu2SnL3), 3c (Ph2SnL3), and 3d (Cy2SnL3) acid were intermediate between square pyramidal and trigonal bipyramidal, and complex 1a (Bu2SnL1) adopted a bipyramidal trigonal geometry (BPT). The sulforhodamine B assay assessed the cytotoxicity of organotin(IV) complexes against the MDA-MB-231 and MCF-7 (human mammary adenocarcinoma) cell lines and one normal COS-7 (African green monkey kidney fibroblast). The IC50 values evidenced a significant antiproliferative effect on cancer cells; the complexes were more potent than the positive cisplatin control and the corresponding ligands, dehydroacetic acid thiosemicarbazone (L1), dehydroacetic acid-N(4)-methylthiosemicarbazone (L2), and dehydroacetic acid-N(4)-phenylthiosemicarbazone (L3). The IC50 values also indicated that the organotin(IV) complexes were more cytotoxic against the triple-negative breast cell line MDA-MB-231 than MCF-7, inducing significant morphological alterations. The interactions of organotin(IV) 1c (Ph2SnL1), 1d (Cy2SnL1), and 1e (((CH3)3SiCH2)2SnL1) were evaluated with ss-DNA by fluorescence; intensity changes of the fluorescence were indicative of the displacement of ethidium bromide (EB), confirming the interaction of the organotin(IV) complexes with ss-DNA; the results showed a DNA binding affinity. The thermodynamic parameters obtained through isothermal titration calorimetry showed that the interaction of 1c (Ph2SnL1), with ss-ADN, was exothermic. Molecular docking studies also demonstrated that the organotin(IV) complexes were intercalated in DNA by conventional hydrogen bonds, carbon-hydrogen bonds, and π-alkyl interactions. These complexes furthermore showed a greater affinity towards DNA than cisplatin.

As a new approach, pyrrolo[1,2-a]pyrazines were synthesized through the cyclization of 2-formylpyrrole-based enaminones in the presence of ammonium acetate. The enaminones were prepared with a straightforward method, reacting the corresponding alkyl 2-(2-formyl-1H-pyrrol-1-yl)acetates, 2-(2-formyl-1H-pyrrol-1-yl)acetonitrile, and 2-(2-formyl-1H-pyrrol-1-yl)acetophenones with DMFDMA. Analogous enaminones elaborated from alkyl (E)-3-(1H-pyrrol-2-yl)acrylates were treated with a Lewis acid to afford indolizines. The antifungal activity of the series of substituted pyrroles, pyrrole-based enaminones, pyrrolo[1,2-a]pyrazines, and indolizines was evaluated on six Candida spp., including two multidrug-resistant ones. Compared to the reference drugs, most test compounds produced a more robust antifungal effect. Docking analysis suggests that the inhibition of yeast growth was probably mediated by the interaction of the compounds with the catalytic site of HMGR of the Candida species.

Abstract 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is a crucial enzyme in the ergosterol biosynthesis pathway. The aim of this study was to obtain, purify, characterize, and overexpress five point mutations in highly conserved regions of the catalytic domain of Candida glabrata HMGR (CgHMGR) to explore the function of key amino acid residues in enzymatic activity. Glutamic acid (Glu) was substituted by glutamine in the E680Q mutant (at the dimerization site), Glu by glutamine in E711Q (at the substrate binding site), aspartic acid by alanine in D805A, and methionine by arginine in M807R (the latter two at the cofactor binding site). A double mutation, E680Q-M807R, was included. Regarding recombinant and wild-type CgHMGR, in vitro enzymatic activity was significantly lower for the former, as was the in silico binding energy of simvastatin, alpha-asarone and the HMG-CoA substrate. E711Q displayed the lowest enzymatic activity and binding energy, suggesting the importance of Glu711 (in the substrate binding site). The double mutant CgHMGR E680Q-M807R exhibited the second lowest enzymatic activity. Based on the values of the kinetic parameters K M and V max , the mutated amino acids appear to participate in binding. The current findings provide insights into the role of residues in the catalytic site of CgHMGR.

Autophagy (macroautophagy) is a survival and virulence mechanism of different eukaryotic pathogens. Autophagosomes sequester cytosolic material and organelles, then fuse with or enter into the vacuole or lysosome (the lytic compartment of most fungal/plant cells and many animal cells, respectively). Subsequent degradation of cargoes delivered to the vacuole via autophagy and endocytosis maintains cellular homeostasis and survival in conditions of stress, cellular differentiation, and development. PrA and PrB are vacuolar aspartyl and serine endoproteases, respectively, that participate in the autophagy of fungi and contribute to the pathogenicity of phytopathogens. Whereas the levels of vacuolar proteases are regulated by the expression of the genes encoding them (e.g., PEP4 for PrA and PRB1 for PrB), their activity is governed by endogenous inhibitors. The aim of the current contribution is to review the main characteristics, regulation, and role of vacuolar soluble endoproteases and Atg proteins in the process of autophagy and the pathogenesis of three fungal phytopathogens: Ustilago maydis, Magnaporthe oryzae, and Alternaria alternata. Aspartyl and serine proteases are known to participate in autophagy in these fungi by degrading autophagic bodies. However, the gene responsible for encoding the vacuolar serine protease of U. maydis has yet to be identified. Based on in silico analysis, this U. maydis gene is proposed to be orthologous to the Saccharomyces cerevisiae genes PRB1 and PBI2, known to encode the principal protease involved in the degradation of autophagic bodies and its inhibitor, respectively. In fungi that interact with plants, whether phytopathogenic or mycorrhizal, autophagy is a conserved cellular degradation process regulated through the TOR, PKA, and SNF1 pathways by ATG proteins and vacuolar proteases. Autophagy plays a preponderant role in the recycling of cell components as well as in the fungus-plant interaction.

Current antidiabetic drugs have severe side effects, which may be minimized by new selective molecules that strongly inhibit α-glucosidase and weakly inhibit α-amylase. We have synthesized novel alkoxy-substituted xanthones and imidazole-substituted xanthones and have evaluated them for their in silico and in vitro α-glucosidase and α-amylase inhibition activity. Compounds 6c, 6e, and 9b promoted higher α-glucosidase inhibition (IC50 = 16.0, 12.8, and 4.0 µM, respectively) and lower α-amylase inhibition (IC50 = 76.7, 68.1, and >200 µM, respectively) compared to acarbose (IC50 = 306.7 µM for α-glucosidase and 20.0 µM for α-amylase). Contrarily, derivatives 10c and 10f showed higher α-amylase inhibition (IC50 = 5.4 and 8.7 µM, respectively) and lower α-glucosidase inhibition (IC50 = 232.7 and 145.2 µM, respectively). According to the structure–activity relationship, attaching 4-bromobutoxy or 4′-chlorophenylacetophenone moieties to the 2-hydroxy group of xanthone provides higher α-glucosidase inhibition and lower α-amylase inhibition. In silico studies suggest that these scaffolds are key in the activity and interaction of xanthone derivatives. Enzymatic kinetics studies showed that 6c, 9b, and 10c are mainly mixed inhibitors on α-glucosidase and α-amylase. In addition, drug prediction and ADMET studies support that compounds 6c, 9b, and 10c are candidates with antidiabetic potential.

Chromenes are compounds that may be useful for inhibiting topoisomerase and cytochrome, enzymes involved in the growth of cancer and fungal cells, respectively. The aim of this study was to synthesize a series of some novel 2-amino-3-cyano-4-aryl-6,7-methylendioxy-4H-chromenes 4a–o and 2-amino-3-cyano-5,7-dimethoxy-4-aryl-4H-chromenes 6a–h by a three-component reaction, and test these derivatives for anticancer and antifungal activity. Compounds 4a and 4b were more active than cisplatin (9) and topotecan (7) in SK-LU-1 cells, and more active than 9 in PC-3 cells. An evaluation was also made of the series of compounds 4 and 6 as potential antifungal agents against six Candida strains, finding their MIC50 to be less than or equal to that of fluconazole (8). Molecular docking studies are herein reported, for the interaction of 4 and 6 with topoisomerase IB and the active site of CYP51 of Candida spp. Compounds 4a–o and 6a–h interacted in a similar way as 7 with key amino acids of the active site of topoisomerase IB and showed better binding energy than 8 at the active site of CYP51. Hence, 4a–o and 6a–h are good candidates for further research, having demonstrated their dual inhibition of enzymes that participate in the growth of cancer and fungal cells.

Introduction: Melatonin is a hormone tightly related to circadian synchronization. Blood levels of melatonin oscillate in 24-h cycles in close accordance with the external illumination. Most circulating melatonin is assumed to be secreted by the pineal gland, whose activity is subordinated to the central circadian clock in the suprachiasmatic nucleus. Pineal melatonin is produced during the dark phase of the endogenous circadian cycle, and acts as a synchronizing signal for secondary oscillators in peripheral tissues. It is recognized that tissues other than the pineal gland synthesize melatonin, but the role of these alternative sources in the circadian melatonin circulation is not clear. The intestine has been shown to contain great amounts of melatonin, estimated to be several-fold larger than that of pineal. However, the origin of intestinal melatonin is debated, for there seem to be several sources besides local synthesis from which melatonin could be incorporated into the intestinal mucosa. Moreover, the relationship of this local melatonin with the pineal circadian activity is obscure. Objective: To describe the relationship between the circadian rhythm with the intestinal melatonin-synthesizing enzymes Material and Methods: In this work we analyzed the presence of the main protein controlling melatonin synthesis, the enzyme aralkylamine N-acetyltransferase (AANAT) in the main different portions of intestine and the changes in its relative concentration along a 24-h cycle. Results: We found that AANAT is present along the intestine with higher levels in the proximal portions. The concentration of AANAT in tissue lysates shows circadian oscillation that roughly parallels that of the pineal with a phase delay of around 4 h. Conclusions: These results indicate that the intestinal wall produces melatonin with a circadian pattern and suggest that this activity is not related with central melatonin signaling but with local timing. Keywords: circadian rhythm, aralkylamine N-acetyltransferase, melatonin, experimental animal

A series of 3-benzoyl imidazo[1,2-a]pyrimidines, obtained from N-heteroarylformamidines in good yields, was tested in silico and in vitro for binding and inhibition of seven Candida species (Candida albicans (ATCC 10231), Candida dubliniensis (CD36), Candida glabrata (CBS138), Candida guilliermondii (ATCC 6260), Candida kefyr, Candida krusei (ATCC 6358) and Candida tropicalis (MYA-3404)). To predict binding mode and energy, each compound was docked in the active site of the lanosterol 14α-demethylase enzyme (CYP51), essential for fungal growth of Candida species. Antimycotic activity was evaluated as the 50% minimum inhibitory concentration (MIC50) for the test compounds and two reference drugs, ketoconazole and fluconazole. All test compounds had a better binding energy (range: −6.11 to −9.43 kcal/mol) than that found for the reference drugs (range: 48.93 to −6.16 kcal/mol). In general, the test compounds showed greater inhibitory activity of yeast growth than the reference drugs. Compounds 4j and 4f were the most active, indicating an important role in biological activity for the benzene ring with electron-withdrawing substituents. These compounds show the best MIC50 against C. guilliermondii and C. glabrata, respectively. The current findings suggest that the 3-benzoyl imidazo[1,2-a]pyrimidine derivatives, herein synthesized by an accessible methodology, are potential antifungal drugs.