Insight to the antifungal properties of Amaryllidaceae constituents
Abstract
Background
The pervasive issue of fungal pathogenesis continues to exert a significant and escalating burden on healthcare infrastructures across both developed and developing nations globally. This concerning scenario is largely exacerbated by two critical factors: first, the gradual and seemingly irreversible decline in the efficacies of existing antifungal medicines over time; and second, the alarming emergence and widespread dissemination of drug-resistant fungal strains. These combined challenges highlight a severe and pressing void in our current therapeutic arsenal against fungal infections. Consequently, there is an urgent and imperative need to discover and develop novel pharmaceutical agents. These new drugs must originate from diverse structural backgrounds, possess significantly improved potencies, and, crucially, exhibit novel modes of action. Such innovations are essential either to fortify current antifungal treatment regimens, enhancing their effectiveness, or to entirely replace contemporary antifungal schedules that are increasingly losing their clinical utility.
Aim
Within this context of urgent need, alkaloids derived from plants belonging to the Amaryllidaceae family have garnered considerable attention due to their demonstrated capacity to exhibit potent growth inhibitory activities against a wide spectrum of fungal pathogens. The primary aim of this comprehensive review is to delve deeply into the mechanistic aspects underlying these observed antifungal activities. It seeks to achieve this by meticulously highlighting the specific molecular targets within fungal cells that these alkaloids interact with, as well as by identifying and discussing the crucial structural features of Amaryllidaceae constituents that serve to enhance their antifungal action. By elucidating these intricate details, the review aims to provide a clearer understanding of how these natural compounds exert their therapeutic effects.
Methods
During the crucial information gathering and compilation stage of this review, extensive and systematic use was made of three prominent and highly regarded scientific database platforms: Google Scholar, SciFinder, and Scopus. These platforms were instrumental in identifying relevant peer-reviewed articles, research papers, and reviews. In the vast majority of instances, direct access to the full-text articles was readily available through journals to which the University of KwaZulu-Natal held institutional licenses. For those instances where such proprietary agreements were not in place, the respective corresponding authors of the papers were directly contacted. This proactive approach ensured that copies of the necessary publications were obtained, thereby maximizing the completeness and accuracy of the gathered information for the review.
Results
The extensive literature search and analysis revealed that while several classes of molecules originating from the Amaryllidaceae plant family have been systematically investigated for their potential antifungal effects, it is primarily two key constituents, namely lycorine and narciclasine, which have collectively provided the most profound and actionable mechanistic insights into their antifungal properties. The mechanistic findings associated with these compounds can be comprehensively summarized across several crucial points: (i) their discernible effects on the structural integrity of the fungal cell wall and cell membrane, which are vital for fungal survival; (ii) their significant influence on fungal morphology, specifically observed in the inhibition of budding processes in yeasts and the disruption of hyphal growth in filamentous fungi; (iii) their direct impact on critical fungal organelles, particularly ribosomes, which are essential for protein synthesis; (iv) their inhibitory effects on the synthesis and integrity of fundamental macromolecules such as DNA, RNA, and proteins, all of which are indispensable for fungal proliferation; and (v) the successful identification of the specific active sites within fungal cells where these Amaryllidaceae constituents exert their inhibitory actions.
Conclusion
In conclusion, the overarching and most significant feature characterizing the antifungal effects of Amaryllidaceae alkaloids, particularly lycorine and narciclasine, is their potent inhibition of protein synthesis within fungal cells. This crucial mechanism involves the specific inhibition of peptide bond formation, a fundamental step in protein synthesis, achieved through their direct binding to yeast ribosomes, predominantly interacting with the 60S ribosomal subunit. Furthermore, related inhibitory effects were observed on the synthesis of both DNA and RNA, indicating a broader impact on fungal macromolecular processes. These adverse biochemical and molecular effects were visibly reflected in significant morphological changes observed on both the fungal cell wall and the cell membrane, underscoring the comprehensive assault these alkaloids launch on fungal cellular integrity. Such detailed observations and mechanistic insights should prove immensely useful and pave the way in the chemotherapeutic arena, particularly if future efforts and resources are strategically shifted towards the targeted development of these Amaryllidaceae alkaloids as promising clinical candidates for new antifungal therapies.
Introduction
Fungal diseases collectively represent a substantial and ongoing global health crisis, impacting an estimated one billion individuals worldwide and tragically accounting for nearly two million deaths annually. These mortality figures are strikingly comparable to those attributed to tuberculosis, and notably, they are more than three times higher than the estimated deaths caused by malaria. Among the myriad fungal-borne diseases, chronic pulmonary aspergillosis, cryptococcal meningitis, disseminated histoplasmosis, fungal keratitis, and *Pneumocystis* pneumonia are currently recognized as posing the greatest concern within public healthcare systems.
The populations most vulnerable to these insidious fungal infections are diverse and often immunocompromised. This includes individuals suffering from cancer, those living with HIV/AIDS, hospitalized patients, individuals with weakened immune systems due to various conditions, as well as recipients of organ transplants and stem cell transplants. Beyond individual susceptibility, broader societal factors have also played a significant role in the recent surge of fungal diseases. These factors encompass increasing globalization and urbanization, the diminishing efficacies of conventional antifungal drugs, and the alarming rise of multidrug resistance among fungal pathogens.
For several decades, synthetic antifungal drugs such as fluconazole, ketoconazole, and itraconazole have demonstrated considerable success in combating fungal infections. However, it is the natural product-based antifungal entities, primarily originating from microorganisms, such as amphotericin B and nystatin, that have maintained an enduring and critical presence in the clinical landscape. Given the diminishing effectiveness of existing treatments, the identification and successful implementation of new antifungal therapeutic regimens are now deeply rooted in collaborative drug discovery efforts spanning various scientific disciplines across both the private and public sectors. In this endeavor, plants have historically offered and continue to provide an exceptionally resourceful platform. Their vast chemical diversity, immense numbers, and long-standing presence in traditional medicinal approaches to fungal diseases make them invaluable sources for novel antifungal compounds.
The efficacy of antifungal agents is intricately linked to their specific modes of action, which are fundamentally determined by the molecular sites they target within fungal cells to exert their inhibitory activities. For instance, the azole, polyene, and allylamine classes of antifungal drugs primarily target the fungal cell membrane, acting either as ergosterol binders or inhibitors of its synthesis. Ergosterol is the principal sterol embedded within the cell membrane of fungi and protozoa, playing crucial regulatory roles concerning membrane asymmetry, fluidity, and overall integrity. The physical binding of ergosterol by polyenes like amphotericin B and nystatin leads to the formation of a polar pore within the fungal cell membrane. This pore allows the uncontrolled seepage of essential charged ions, such as hydrogen (H+) and potassium (K+), as well as other vital intracellular molecules, ultimately culminating in devastating cellular disruption and fungal cell death.
In contrast, azole antifungals, including clotrimazole, itraconazole, and miconazole, function as inhibitors of 14α-demethylase, an enzyme critically required for the metabolic pathway that synthesizes ergosterol from lanosterol. Similarly, allylamines such as terbinafine exert their antifungal effects by inhibiting squalene epoxidase, an enzyme crucial for the conversion of squalene to lanosterol. This inhibition consequently blocks the synthesis of ergosterol, disrupting membrane integrity. A third distinct group of antifungal agents, exemplified by echinocandins such as anidulafungin, caspofungin, and micafungin, specifically target the fungal cell wall. They inhibit the synthesis of cell wall glucan through the non-competitive inhibition of 3-β-glucan synthase, an enzyme vital for maintaining the structural integrity of the fungal cell wall.
Finally, another significant group of antifungal agents, represented by compounds like flucytosine and griseofulvin, manifest their action by engaging fungi at the intracellular level, interfering with fundamental cellular processes. Flucytosine, for example, is metabolized within fungal cells first into the cytostatic fluorouracil, and subsequently into 5-fluorouridinetriphosphate. This derivative then aberrantly participates in RNA biosynthesis, thereby disrupting the accumulation of several essential proteins necessary for fungal survival and proliferation. Additionally, flucytosine can also be converted to 5-fluorodeoxyuridinemonophosphate, a metabolite capable of inhibiting fungal DNA synthesis. Griseofulvin, on the other hand, exerts its antifungal effect by binding to tubulin, a protein essential for the formation of microtubules. This binding disrupts microtubule function, thereby inhibiting critical cellular processes like mitosis. Furthermore, griseofulvin has the unique ability to bind to keratin within its precursor cells, rendering them resistant to subsequent fungal attacks.
The plant family Amaryllidaceae holds a prominent position in traditional and alternative medicine, where many of its members are historically employed to treat various ailments, a significant number of which are believed to be consequences of fungal pathogenesis. To date, close to thirty distinct species, spanning twelve different genera within the Amaryllidaceae family, have been rigorously examined for their antifungal effects against approximately twenty different fungal pathogens. The detection of good extract activities, often at concentrations in the microgram per milliliter range, has served as a powerful impetus for concerted efforts to identify the specific active constituents responsible for these effects and to elucidate their precise modes of action. To date, approximately forty distinct constituents isolated from this plant family have been tested against nearly fifty different fungal pathogens. Encouragingly, many of these constituents have demonstrated significant growth inhibitory activities against a number of targets, with the lowest observed minimum inhibitory concentration (MIC) recorded at an impressive 4 micrograms per milliliter. This review specifically focuses on studies that have provided crucial insights into the molecular and structural basis of the antifungal action of Amaryllidaceae compounds, with a particular emphasis on its isoquinoline alkaloids, which constitute the majority of its rich phytochemical principles.
Sites of Action
Despite the extensive investigation into the antifungal properties of Amaryllidaceae alkaloids, there remains, in general, a noticeable scarcity of detailed information specifically delving into the precise cellular and molecular sites targeted by these compounds to bring about their antifungal effects. However, a substantial amount of research has been successfully undertaken to elucidate their protein-inhibitory effects at the intracellular level, as well as their allied interactions with fungal ribosomes. These findings will be discussed in depth in the succeeding sections of this manuscript.
While no single Amaryllidaceae constituent has been directly examined for its effects on the fungal cell wall *per se*, recent findings from the screening of crude extracts of *Hymenocallis littoralis* in *Candida albicans* cultures strongly suggest that these compounds may indeed be acting at the level of this crucial morphological feature. Specifically, a methanol-chloroform (3:1) leaf extract of *H. littoralis* was shown to exhibit weak, yet discernible, growth inhibitory effects on *C. albicans*, with a minimum inhibitory concentration (MIC) of 6.25 milligrams per milliliter. Follow-up time-kill experiments provided further intriguing details: at half the MIC, *C. albicans* cells remarkably maintained their vibrancy throughout the maximum tested time period of 32 hours, although by this point, the number of viable cells had reduced to approximately 50% of that observed in control cells. In contrast, at the higher tested dosages of 6.25 and 12.5 milligrams per milliliter, the extract initially demonstrated a cytostatic effect up to the 20-hour mark, after which it became visibly fungicidal. This fungicidal effect was particularly striking at twice the MIC (12.5 milligrams per milliliter), where the majority of cells were rapidly depleted within just 8 hours.
Scanning electron microscopy (SEM) observations, conducted in parallel with the time-kill experiments, proved to be highly insightful. At the 0-hour mark, prior to treatment, *C. albicans* cells maintained their characteristic oval shape and possessed a smooth, intact cell wall. The budding stage, indicative of active proliferation, was also clearly visible during observations at this time point. However, following 8 hours of treatment with the extract, the fungal cell walls exhibited a markedly coarse and irregular appearance. After 16 hours, the cells became noticeably distorted, characterized by prominent convolutions and invaginations across their surface. By the 20-hour mark, the collapse of the cell walls became an undeniable and striking feature in the captured micrographs. Several isoquinoline alkaloids, including the two primary constituents lycorine (1) and narciclasine (2), have been previously identified in the ‘beach spider lily’ (*H. littoralis*). These compounds may well explain the observed cell wall injurious effects of the plant extract against *C. albicans*. This possibility is particularly intriguing given that the majority of studies conducted thus far to investigate the antifungal modes of action of Amaryllidaceae alkaloids, as will be further attested below, have primarily focused on these two specific alkaloids. Furthermore, it is also conceivable that the various alkaloid extractives present in *H. littoralis* may be operating synergistically to collectively effect their damaging action on the *C. albicans* cell wall.
Lycorine was also extensively screened against twenty-four different crop pathogenic fungi, where it consistently exhibited good growth inhibitory ratios. Notably, strong activity was observed against *Fusarium graminearum* at tested dosages of both 500 micrograms per milliliter (demonstrating 74.7% inhibition) and 100 micrograms per milliliter (showing 63.7% inhibition). Due to this pronounced activity, *F. graminearum* was subsequently selected as the target organism for a deeper probe into the underlying mechanistic effects of lycorine. The natural reddish tint of the mycelia in *F. graminearum* proved advantageous, as it facilitated convenient observations of its microstructure and enabled accurate measurements of its growth and development. Additionally, the characteristic cluster formation observed in the mycelial growth of *F. graminearum* allowed for convenient extraction and subsequent experimentation.
Microscopic examinations, conducted at both 10x and 40x magnification, were performed on mycelia obtained from *F. graminearum* cells that had been treated with lycorine at its minimum inhibitory concentration (MIC) of 50 micrograms per milliliter. These observations revealed distinct differences: the control groups, under both magnifications, exhibited a significantly greater mycelial density and more extensive branching compared to the cells that had been treated with lycorine, indicating an inhibitory effect on fungal growth. The effects of lycorine on the fungal cell membrane were also investigated using mycelia from cells supplemented with 50 micrograms per milliliter lycorine. Cellular permeability, a critical indicator of membrane integrity, was assessed by measuring conductivity at designated time intervals of 0, 2, 4, 6, 8, and 10 hours. These measurements consistently showed that the differences in conductivity between the control and lycorine-treated groups gradually increased over the 10-hour period. This rising conductivity strongly indicates that lycorine is capable of disrupting the cytomembrane structure, leading to the undesirable exosmosis, or leakage, of essential intracellular materials.
Further evidence supporting lycorine’s influence on *F. graminearum* cellular metabolism was garnered from the observed reduction in succinate dehydrogenase (SD) activity within the mycelia of lycorine-treated cells. SD is an enzyme that plays a vital role in both the citric acid cycle and electron transport chain, making its activity a reliable indicator of the overall vibrancy and metabolic health of fungal cells. The soluble protein content of both the test and control groups exhibited a gradual decrease up to the 48-hour interval. However, after this point, an interesting increase in soluble protein content was observed specifically in the lycorine-treated group of cells. This increase is largely attributable to the leakage of intracellular material, including proteins, through the cell membrane damage induced by lycorine. Conversely, the later increase in protein content of the control group after 72 hours was suggested to be a consequence of increased mycelial shrinkage and cell death occurring naturally in crowded, aged cultures.
Morphological Effects
Morphological investigations provided further insights into the effects of lycorine (1) on fungal cells. Specifically, lycorine’s ability to inhibit the critical transition from the blastospore to the hyphal state was examined in *Candida albicans* cultures. This pathogenic yeast is well-known for its morphological plasticity, capable of growing in multiple distinct forms, including the blastospore (yeast-like), pseudohyphal, and true hyphal states. Its virulence, to a considerable extent, is dependent on its inherent ability to interconvert between these morphological states, a process meticulously regulated by various cellular and environmental factors.
In a comprehensive assay involving 480 molecules from the ICCB collection at Harvard University, lycorine was one of fifty-three compounds that demonstrated cytotoxic effects on a clinical isolate of *C. albicans* (strain SC5314). The testing was conducted at 37°C on Spider medium, utilizing a concentration of 130 micromolar (µM) over a period of 4 hours. The primary objective of this assay was to identify molecules capable of suppressing hyphal formation, such that only blastospores would be observable in the microtiter well plates. While lycorine inhibited the morphological changes, subsequent analysis revealed that its detrimental effects in this context were primarily a consequence of general cytotoxicity, rather than a specific inhibition of the blastospore to hyphal transition process itself.
In contrast to these findings, earlier and seemingly unrelated studies provided evidence that lycorine actually acted as a stimulator of budding in *Cryptococcus terreus*. In these experiments, the number of *C. terreus* cells cultured on YNB liquid medium was observed to significantly increase in the presence of lycorine at a concentration of 100 µM. Scanning electron microscopy (SEM) micrographs further revealed notable morphological changes: cells cultured in medium alone typically had mean dimensions of 9.5 x 7.8 µm, whereas cells treated with lycorine were distinctly smaller and more rounded, measuring 7.0 x 6.5 µm on average. By comparison, cultures of *Cryptococcus dimennae* supplemented with the same concentration of lycorine showed negligible effects on cell numbers. Furthermore, *C. dimennae* cells cultured both in the absence and presence of lycorine maintained similar mean dimensions of 6.0 x 3.0 µm. Nonetheless, it was an interesting observation that the lycorine-containing medium in which *C. terreus* had been cultivated was capable of stimulating the growth of *C. dimennae* cells. In *C. dimennae*, as in *C. terreus*, the overall increase in cell number was observed to be greater than the weight increase of dry cultures. This discrepancy suggested that while lycorine alone might not directly affect *C. dimennae*, it could do so presumably after undergoing some form of modification or transformation by *C. terreus*.
Lycorine
Lycorine alkaloids constitute a large and continuously expanding group of natural compounds found within the Amaryllidaceae family. These compounds are particularly renowned for their intriguing antiproliferative effects. To date, seven representatives from this group have been specifically examined for their antifungal properties, with the parent compound, lycorine (1), consistently demonstrating the best general activity across seven different fungal strains. An impressively low IC50 value of 6.3 micrograms per milliliter was calculated, specifically pertaining to observations made in *Candida candida* cells. Intriguingly, antifungal effects were among the earliest biological properties demonstrated for Amaryllidaceae constituents in general, and for lycorine alkaloids in particular.
Early research demonstrated that lycorine was a potent inhibitor of cell division in liquid cultures of *Saccharomyces cerevisiae*. A clear dose-dependent effect on cell division was observed, characterized by an inverse relationship between the quantity of lycorine used and the corresponding number of viable *S. cerevisiae* cells. For instance, following treatment with lycorine at concentrations of 0, 0.01, 0.025, 0.05, and 0.1 mM, the corresponding cell counts (x 105) were 87.2, 64.6, 28.7, 8.7, and 6.1, respectively, illustrating a dramatic reduction in cell numbers with increasing lycorine concentration. Cytological analyses further indicated that these inhibitory effects on cell division corresponded with a greater accumulation of cells during the interphase period of the cell division cycle. A further interesting observation made in this study was that at concentrations exceeding 10 mM, lycorine produced notable cell damage and necrotic effects. Such cytotoxic effects have since been recognized as integral to its broader antiproliferative properties, including those observed in various cancer cells. Given that both RNA and protein synthesis are fundamental processes required for cell division and elongation, it was initially suggested that lycorine could potentially be an inhibitor of either or both of these essential processes.
Building upon these initial findings, the same authors conducted further studies to establish lycorine’s response in metabolic processes directly related to cell division. To this effect, it was demonstrated that following treatment with lycorine (at 0.1 mM) in *S. cerevisiae* cells, the amounts of 14C-leucine incorporated into protein and 3H-uridine incorporated into RNA both decreased. However, time course experiments provided a crucial distinction: the dramatic drop in 14C-leucine incorporated into protein occurred prior to the inhibitory effect of lycorine on 3H-uridine incorporation into RNA. Furthermore, *in vitro* experiments conducted in a cell-free system revealed that the elongation of polypeptide chains in *S. cerevisiae* ribosomes was not suppressed by lycorine, even at concentrations up to 1 mM.
Del Giudice and colleagues carried out more extensive studies of lycorine, utilizing several strains of *S. cerevisiae*, including the rho0, rho+, rho-, and mit- strains. From these experiments, it was clearly demonstrated that lycorine, at three tested concentrations (50, 150, and 250 µg/mL), effectively inhibited the growth of the rho+, rho-, and mit- strains. However, significantly, it did not inhibit the rho0 strain, which is characterized by a complete absence of mitochondrial DNA. Interestingly, it was also shown that the inhibitory effect of lycorine on the growth of the rho+ *S. cerevisiae* strain could be remarkably counteracted by the inclusion of ascorbic acid in the plating medium. This is a particularly insightful observation, as lycorine is well-established as a potent inhibitor of ascorbic acid biosynthesis in plants. In a related context, regarding the inhibitory effect of lycorine on ascorbic acid biosynthesis in fungal cultures, Onofri and colleagues demonstrated that the growth of two strains (*C1* and *C3*) of *Cryptococcus laurentii*, which were isolated from the root tips of the Amaryllidaceae plant *Narcissus pseudonarcissus*, was significantly inhibited by lycorine at 100 µM. A further compelling finding from this study was that the *in vitro* production of ascorbic acid from L-galactonic acid-γ-lactone in cultures of both *C1* and *C3* strains was also inhibited in the presence of lycorine. By contrast, the *C. laurentii* strain *C4* was not inhibited by lycorine and notably did not produce ascorbic acid when cultivated either with or without L-galactonic acid-γ-lactone.
While both cytosolic and mitochondrial protein synthesis were only slightly inhibited in *S. cerevisiae* cultures, the rho- strains displayed particular sensitivity to lycorine, despite their known deficiency in mitochondrial protein synthesis. From these observations, it was suggested that these characteristics of lycorine might be manifesting via an indirect effect on mitochondrial DNA replication or transcription. Separate studies by Karadeniz and colleagues later confirmed that lycorine was indeed capable of interacting with eukaryotic DNA. To this effect, differential pulse voltammetry (DPV) analysis revealed that signals originating from guanine and adenine residues were markedly diminished as a consequence of lycorine’s interaction with calf thymus DNA. However, it has been pointed out that due to its non-planar molecular structure, lycorine (1) must bind to calf thymus DNA in a manner other than through direct intercalation between base pairs. In contrast, lycorine was shown to be capable of intercalating with *Saccharomyces* tRNA, presumably via its flexible double-stranded fragments.
It has also been observed that lycorine (1), dihydrolycorine (3), and pseudolycorine (4) exhibited dose-dependent inhibitory effects on protein synthesis in Krebs II ascites cells. Concurrently, these alkaloids also slowed down DNA synthesis, but notably had little effect on RNA synthesis. The dose range at which protein synthesis was inhibited by these three alkaloids coincided remarkably with their minimum growth inhibitory concentrations observed in HeLa human cervical adenocarcinoma cells (6, 100, and 25 µM, respectively). Such effects have been shown to be closely associated with compounds known to be potent inhibitors of protein synthesis in eukaryotic cells.
Further mechanistic insight into the protein inhibitory effects of lycorine was provided through binding experiments conducted with wheat germ ribosomes. From these experiments, it was suggested that a change in the conformation of the 60S ribosomal subunit in the region of the peptidyl transferase center (PTC), induced by lycorine binding, could impede oligonucleotide donor adsorption. Earlier research had indicated that since lycorine can prevent the transfer of the N-acetylleucine residue from CACCA-Leu-Ac (the 3’-terminal fragment of AcLeu-tRNA) onto puromycin, it might be capable of inhibiting the transpeptidation reaction. However, the findings of Kukhanova and colleagues indicated to the contrary that lycorine did not influence the reaction of PA-Met-f [3’(2’)-O-(N-formylmethionyl)-adenosine-5’-phosphate] with CACCA-Phe (the 3’-terminal fragment of Phe-tRNA) in the presence of cytidine-5’-phosphate. Nonetheless, the selective inhibition of the binding of the pentanucleotide donor substrate CACCA-Leu-Ac to the donor site of the PTC was a distinct characteristic of lycorine’s interaction with wheat germ ribosomes. The effect of lycorine on viral protein synthesis has also been investigated in poliovirus-infected HeLa cells. From this study, it was shown that lycorine dose-dependently suppressed the incorporation of [3H]leucine, although it never completely halted overall translation. A further suggestion made in this context was that lycorine might be acting specifically at the stage of chain termination.
As a direct continuation and follow-up to the groundbreaking work of Del Giudice and colleagues on the rho0, rho+, rho-, and mit- strains of *S. cerevisiae*, Massardo and colleagues selected suppressive rho- mutants, which exhibited similar sensitivities to lycorine as the wild-type strain, as substrates for further mechanistic probes. In these subsequent investigations, hypersuppressive petites were demonstrated to be nearly as resistant as rho0 mutants. Conversely, the isogenic rho- petites, which had retained larger segments of the mitochondrial genome, proved to be sensitive to lycorine. The same authors subsequently provided conclusive evidence strongly supporting the ability of lycorine (1) to differentiate between cells that possess defective mitochondrial DNA (mit- cells) and those with a complete deficiency of this crucial molecule (rho0 cells). As such, rho0 cells were consistently shown to be resistant to lycorine, while both mit- and wild-type cells exhibited similar sensitivities.
In an extension of these concerted efforts, Del Giudice and colleagues further demonstrated that the extent of suppressiveness in rho2 mutant cells directly correlated to their degree of resistance to lycorine. This finding highlighted a critical point: resistance to lycorine is not solely influenced by the overall mitochondrial status (encompassing rho1, mit-, rho-, rho0 strains), but is also significantly modulated by the specific gene sequences that are retained and subsequently amplified in the hypersuppressive petite variants. The final piece of research addressing this theme was undertaken by Massardo and colleagues, wherein it was definitively proved that resistance to lycorine provides a reliable method to ascertain the mitotic stability of rho+ and rho- mitochondrial genomes, both in the presence and absence of CCE1 gene products. The specific endonuclease CCE1 is known for its crucial role in resolving recombination intermediates within mitochondrial DNA. In their study, samples of various rho+ and rho- mitochondrial genomes were meticulously evaluated using both lycorine resistance assays and DAPI (4′,6-diamidino-2-phenylindole) staining. To this extent, higher levels of rho0 cells were consistently observable in nearly all instances when the signature CCE1 gene product was absent. A notable exception to this observation was that those rho- genomes composed entirely of 100% A-T base pairs were not influenced by the absence of CCE1, suggesting a unique resilience or an alternative mechanism of stability in these particular mitochondrial genomes.
Narciclasine
Narciclasine is a representative alkaloid belonging to the phenanthridone class of Amaryllidaceae compounds. Its biological properties have been most extensively investigated in the context of cancer research, where it has shown significant promise. However, studies specifically addressing its antifungal activities have been more limited, primarily focusing on two key constituents: narciclasine (2) itself and *trans*-dihydrolycoricidine (5). Despite this limitation, both compounds have demonstrated good antifungal activities against five different fungal pathogens. Notably, *Cryptococcus neoformans* proved to be particularly susceptible to treatments with concentrations ranging from 8 to 32 micrograms per milliliter.
Similar to the mechanistic studies conducted for lycorine, the research into narciclasine’s antifungal mechanisms has primarily utilized the yeast *Saccharomyces cerevisiae* as a model organism. The earliest of these studies revealed that narciclasine effectively inhibits protein synthesis in yeast ribosomes by specifically blocking peptide bond formation. Further investigations elucidated that narciclasine achieves this by binding directly to the 60S subunit of yeast ribosomes. This binding impedes peptide bond formation by sterically hindering the attachment of the donor substrate (via its 3′-terminus) to the peptidyl transferase center (PTC) of the ribosome. It was also demonstrated that narciclasine strongly inhibited the ribosomal binding of [3H]anisomycin, a known inhibitor of peptide bond formation, but was a poor inhibitor of [14C]trichodermin binding. This differential inhibition indicated that narciclasine and anisomycin likely share a common binding site within the PTC. Both anisomycin and trichodermin are recognized inhibitors of peptide bond formation in eukaryotic ribosomes, where they also play significant roles in substrate binding.
As a crucial extension of these foundational findings, narciclasine (2) was also examined for its activity against two specific strains of *S. cerevisiae*: the mutant anisomycin/trichodermin resistant TR1 strain and the wild-type haploid strain Y166. These experiments clearly demonstrated that any alteration within the PTC of the 60S ribosomal subunit in the mutant strain was accompanied by a clear resistance to narciclasine. Correspondingly, the protein fraction derived from wild-type cells showed a dose-dependent decline in [3H]leucine incorporation following treatment with narciclasine, indicating inhibited protein synthesis. In stark contrast, under similar conditions in mutant cells, the uptake of the amino acid was not halted, consistent with their resistance. Furthermore, it was revealed that polypeptide synthesis occurring on ribosomes or polysomes was more significantly affected by narciclasine in wild-type Y166 cells than in the mutant TR1 cells. This compelling evidence strongly indicated that an alteration in the mutant ribosome was directly responsible for the observed resistance to narciclasine.
To pinpoint the precise location of narciclasine resistance within the ribosomal structure, reassembled hybrid ribosomes, constructed from subunits derived from both the wild-type and mutant strains, were utilized. From this meticulous analysis, it unequivocally emerged that the 40S ribosomal subunit was unsuitable as a location for such action. This finding strongly suggested that the mutation responsible for narciclasine resistance must be expressed via the 60S ribosomal subunit. Subsequently, known peptidyl transferase inhibitors, including anisomycin and chloramphenicol (amongst other antibiotics), along with narciclasine, became the focal point of studies aimed at probing their binding modes in ribosomes derived from *Haloferax mediterranei*, *Escherichia coli*, and *S. cerevisiae*. Despite notable similarities in the sequence and secondary structure of the PTC across the 23S-like rRNAs of these diverse organisms, these inhibitors were particularly attractive due to their varying levels of specificity for archaeal, bacterial, and eukaryotic ribosomes. The binding sites were meticulously probed by incubating each inhibitor (at a final concentration of 100 µM) with ribosomes from the three organisms, followed by rigorous chemical analysis of their respective 23S-like rRNAs. The results were noteworthy, indicating subtle changes in one or two nucleotides (for anthelmycin and narciclasine) to more extensive alterations involving up to eight or nine nucleotides (for virginiamycin M1). The inference drawn from these observations was that these transferase inhibitors were capable of inducing and stabilizing a specific, functional conformer of the PTC, thereby disrupting its normal activity.
It is generally acknowledged that structural data concerning ribosomal inhibitors in eukaryotic cells are considerably more limited compared to those targets, primarily antibiotics, that are associated with bacterial systems. With this in mind, crystallographic studies were strategically undertaken to probe such effects, utilizing the 80S ribosome of *S. cerevisiae* as a model. The targets for this investigation comprised twelve eukaryote-specific inhibitors, including lycorine (1) and narciclasine (2), as well as four broad-spectrum inhibitors such as pactamycin and blasticidin S. High-resolution X-ray crystallographic analysis successfully identified four likely binding sites for these inhibitors in complexes with the 80S ribosome. These included the peptidyl transferase center (PTC) and the tRNA E-site on the large 60S subunit, as well as the mRNA channel and the decoding center (DC) on the small 40S subunit. It was definitively demonstrated that all twelve inhibitors targeted both tRNA and mRNA binding sites. However, lycorine (1) and narciclasine (2) were also specifically shown to be associated with the A-site of the PTC. The precise alignment of the aminoacyl-tRNA and peptidyl-tRNA substrates at the A- and P-sites, respectively, of the PTC is an absolute and stringent requirement for the accurate and efficient formation of peptide bonds. It is well-established that following binding, A-site inhibitors typically induce a pattern of similar structural rearrangements that tend to propagate away from the PTC, further disrupting ribosomal function. A distinct feature distinguishing the binding of lycorine (1) and narciclasine (2) is the strikingly different positioning of their A-ring methylenedioxy group compared to other well-known A-site inhibitors, such as the trichothecenes. This structural nuance may contribute to their specific inhibitory profiles.
Considering that the antifungal activities of Amaryllidaceae alkaloids are primarily attributed to their ribosomal-inhibitory functions, observations by Cundliffe and Demain offer a rather fascinating perspective on the topic. They note that antibiotics, which are produced as secondary metabolites by microorganisms, possess inherent potential for autotoxicity, meaning they could be harmful to the producing organism itself. To overcome such threats, these organisms have evolved sophisticated self-protective mechanisms. These mechanisms include: (i) modification of drug receptors, rendering them resistant to the antibiotic; (ii) synthesis of entirely new receptors that are intrinsically drug-resistant; (iii) the employment of metabolic shielding apparatuses that detoxify or sequester the antibiotic; and (iv) the application of efflux strategies, actively pumping the antibiotic out of the cell, thereby preventing or minimizing interactions between the drug and its intracellular target. Such intricate mechanisms have been widely and extensively examined in antibiotic-producing prokaryotes, particularly the Actinomycetes. However, comparatively few examples exist for lower eukaryotes, and the mechanisms in higher organisms largely remain unexplored. Plant alkaloids, which have been variously hypothesized to function as nitrogen excretion channels or even as deterrents against herbivory, are, in essence, natural antibiotics, even if some can be harmful to humans. The authors then cite the example of narciclasine, derived from the bulbs of various *Narcissus* species, which, as outlined previously, can bind to the large subunit of eukaryotic ribosomes, where it potently inhibits peptide bond synthesis. Perplexingly, ribosomes from Amaryllidaceae plants themselves have never been probed for possible resistance to narciclasine or any other alkaloid from the family. Cundliffe and Demain propose that such (or related) effects could offer a plausible explanation for how higher organisms are able to avoid self-suicide, and that a deeper understanding of these intrinsic mechanisms could yield significant underlying benefits for future drug discovery efforts.
Additional valuable information regarding the antifungal attributes of narciclasine (2) was gleaned from studies that focused on its association with the translation elongation factor eEF1A. In this regard, eEF1A was found to be detectably associated with the Rho GTPase protein Rho1p within vacuoles isolated from the cell membrane of *S. cerevisiae* cells. eEF1A, in its active GTP-bound state, plays a pivotal role in the polypeptide chain elongation process by binding to and facilitating the precise delivery of aminoacylated tRNA molecules to the A-site of the ribosome. Conversely, the inactive GDP-bound form of eEF1A is released from the ribosome but can be reactivated by the guanine nucleotide exchange factor eEF1Bα before subsequently binding to another aminoacylated-tRNA molecule. Beyond its critical function during the elongation step of translation, eEF1A is recognized as a truly multi-functional protein, with diverse roles in a variety of other essential cellular processes, including nuclear export and F-actin remodeling. Rho GTPases, on the other hand, function as crucial molecular switches involved in the modulation of numerous cellular processes, most notably those pertaining to actin dynamics. Rho1p, specifically, is a GTP-binding protein that participates in the latter stages of vacuolar fusion. It is also an essential component required for the activation of protein kinase C (Pkc1p) and β(1-3)glucan synthase, as well as for critical processes such as cell polarization and cell cycle progression.
Narciclasine was strategically utilized to probe the role of actin in the vacuolar localization of eEF1A. Narciclasine (2) was chosen as a target drug for this specific purpose because it is known to bind directly to eEF1A, thereby suppressing its actin bundling activity. In line with this, *S. cerevisiae* vacuoles exhibited marked destabilization and a propensity for leakage when exposed to narciclasine at a concentration of 0.5 µM. Since *per se* narciclasine exposure did not attenuate the overall vacuolar association of eEF1A, it was, by extrapolation, presumed that eEF1A vacuole localization did not directly depend upon actin binding. However, it was definitively shown that the submembranous distribution of eEF1A was, in fact, disrupted by narciclasine, and was therefore indeed actin-dependent. Furthermore, it was revealed that the interaction with Rho1p occurred specifically via the eEF1A C-terminal subdomain. Additionally, while eEF1A itself did not directly promote vacuolar fusion, the overexpression of its Rho1p-interacting subdomain had a marked and noticeable effect on vacuolar morphology. From these combined observations, it was proposed that eEF1A binds to Rho1p-GDP on the vacuolar membrane, from where it is subsequently released following the activation of Rho1p. It then associates with actin bundling to stabilize the newly fused vacuoles. Interestingly, it has been previously noted that narciclasine-induced growth impairment of glioblastoma multiforme tumor cells ensued via a depletion of mitotic rates. In this instance, narciclasine, by increasing GTPase RhoA activity, also activated the Rho/Rho kinase/LIM kinase/cofilin signaling pathway, in addition to inducing RhoA-dependent actin stress fiber formation.
Other Alkaloids of the Amaryllidaceae
Beyond lycorine and narciclasine, other alkaloids from the Amaryllidaceae plant family that have been subjected to investigation for their antifungal modes of action include haemanthamine (6) and pretazettine (7). Haemanthamine is a member of the crinane group of Amaryllidaceae alkaloids, which are well-documented for their antiproliferative effects. In contrast, pretazettine is representative of a minor alkaloid group within the family, generally possessing comparatively more limited biological potential. Given that lycorine (1), pseudolycorine (4), haemanthamine (6), and pretazettine (7) share similarities in their chemical structure, biogenesis, and overall biological function, it was hypothesized that they might also share a common mode of action as well as a common binding site within yeast ribosomes.
Nonetheless, experiments demonstrated that ribosomes derived from the narciclasine-resistant mutant TR1 strain of *S. cerevisiae* displayed cross-resistance to both haemanthamine (6) and pretazettine (7), but notably not to lycorine (1) and pseudolycorine (4), across a wide concentration range from 0.1 µM to 1 mM. Interestingly, in the fragment reaction assay, ribosomes from the TR1 mutant strain were found to be more sensitive to lycorine and pseudolycorine than ribosomes from the wild-type Y166 strain. From these contrasting observations, it was surmised that narciclasine, haemanthamine, and pretazettine must share a common binding site on the peptidyl transferase center (PTC) of the 60S ribosomal subunit, a site that is distinct from the binding site utilized by lycorine and pseudolycorine.
Later, Baez and Vasquez demonstrated through their research that the binding of [3H]narciclasine to yeast ribosomes could be inhibited (within the 0.01 to 1 mM range) by several other Amaryllidaceae alkaloids. These included lycorine (1), dihydrolycorine (3), pseudolycorine (4), haemanthamine (6), pretazettine (7), and *trans*-dihydronarciclasine (8). However, the lycorane alkaloids, namely lycorine, dihydrolycorine, and pseudolycorine, exhibited only minor inhibitory effects on [3H]narciclasine binding. In stark contrast, substantial inhibition of binding was observed for haemanthamine and pretazettine. Intriguingly, it is precisely these latter two alkaloids that display cross-resistance to narciclasine (2), reinforcing the notion of a shared binding mechanism. Despite this, none of the six alkaloids tested, even at concentrations as high as 500 times that of [3H]narciclasine, were able to effect complete inhibition of [3H]narciclasine binding. Furthermore, no inhibition of [3H]narciclasine binding was observed in the presence of several other known inhibitors of peptide bond formation, including blasticidin S, gougerotin, puromycin, and sparsomycin. Such competition experiments further indicated that the narciclasine binding site on the 60S ribosomal subunit overlaps with, rather than being identical to, the binding sites for anisomycin, fusarenon X, trichodermin, trichothecin, and verrucarin A. Overall, these results strongly suggested that the respective ribosomal binding sites for the six Amaryllidaceae alkaloids were presumably also overlapped with, rather than identical to, the corresponding binding site for narciclasine.
In a manner similar to studies undertaken on the 80S ribosome of *S. cerevisiae* complexed with narciclasine, the X-ray crystal structure of haemanthamine (6) was determined at a 3.1 Å resolution in conjunction with the same ribosome. This high-resolution structural analysis revealed that haemanthamine binds to the peptidyl transferase center (PTC) on the large ribosomal subunit. It occupies a distinct pocket in the immediate vicinity where the CCA terminus of the tRNA A-site is typically located during peptide chain elongation. Precise spatial measurements further highlighted that only one molecule of haemanthamine can bind per ribosome, which would allow for significant specificity in drug targeting, potentially minimizing off-target effects. While the binding pocket of haemanthamine was found to be very similar to that of narciclasine, a key difference emerged in the specific manner in which each remained bound to the 25S rRNA residue of the A-site pocket within the PTC. Interestingly, structural superposition of the 25S rRNA with the 28S rRNA derived from a human 80S ribosome in complexation with haemanthamine revealed notable similarities. This striking resemblance suggests that it may indeed be possible for haemanthamine to bind to human ribosomes in a similar fashion, an observation with significant implications for potential therapeutic applications in mammals.
Miscellaneous Constituents of the Amaryllidaceae
Given that the alkaloid constituents represent the chief phytochemical principles of the Amaryllidaceae family, there is a general paucity of detailed information available concerning the diverse biological properties of other non-alkaloidal constituents. This scarcity is even more pronounced when it comes to the antifungal properties of such entities. To date, the only non-alkaloidal targets that have been rigorously examined for antifungal activity include two ceramides, three flavonoids, and a single glutamine-rich peptide.
The ceramides candidamide A (9) and candidamide B (10), isolated from *Zephyranthes candida*, both exhibited activity against *Aspergillus niger*, *Candida albicans*, and *Trichophyton rubrum*, with minimum inhibitory concentrations (MICs) ranging from 20 to 50 micrograms per milliliter. Ceramide compounds, such as Myriocin (11), a commercially recognized sphingolipid synthesis inhibitor, are also well-known for their established antifungal properties. Myriocin, for instance, has been specifically shown to be effective in inhibiting biofilm formation in *Aspergillus fumigatus*. It is widely recognized that several bacterial and fungal pathogens secrete complex polymers that serve as the fundamental matrix for biofilm formation. Such microbial accumulations, as observed for example in chronic aspergillosis, are not only unusually resistant to normal host immune responses but also pose a significant additional threat due to their largely impenetrable nature by most conventional antimicrobial drugs.
Among the flavonoids, sideroxylin (12), 6,7-dimethyldenaromadendrin (13), and farrerol (14), all isolated from *Scadoxus pseudocaulus*, exhibited the best overall antifungal activities among all tested non-alkaloidal Amaryllidaceae constituents. Their MICs against cultures of *C. albicans*, *Candida parapsilosis*, and *C. neoformans* ranged from 8 to 128 micrograms per milliliter. The antifungal effects of flavonoid-related structures have been previously associated with a variety of mechanisms, including efflux pump inhibition, prevention of biofilm formation, inhibition of ergosterol biosynthesis, as well as modulation of cell wall binding properties.
Conclusions
In summary, the constituents derived from the Amaryllidaceae plant family have been definitively shown to elicit intriguing and significant antifungal effects at the molecular level. Our analysis encompassed eight representatives from the phenanthridone, lycorane, and tazettine groups of alkaloids, in addition to six non-alkaloidal components. However, it is crucial to note that the most profound and detailed molecular insights into these antifungal effects have been primarily afforded by studies focusing on the two key alkaloids, lycorine and narciclasine. These foundational studies have been meticulously modeled and carried out predominantly on the cells, organelles, and macromolecules of the yeast species *Saccharomyces cerevisiae*, which has served as a critical model organism.
At the macroscopic level, there is compelling evidence to demonstrate that Amaryllidaceae alkaloids possess the ability to target both the cell wall and cell membrane of fungal pathogens, vital structures for fungal integrity and survival. Furthermore, these compounds can significantly affect the overall size, shape, and texture of fungal cells, and importantly, they can impact other crucial morphological features such as budding in yeasts and hyphal growth in filamentous fungi. Both narciclasine and lycorine have been explicitly shown to inhibit cell division, as well as the fundamental processes of RNA synthesis and DNA replication within *S. cerevisiae* cells. Markedly potent inhibitory effects were also consistently observed on protein synthesis following exposure to these alkaloids, ultimately leading to significant cellular damage and eventual fungal cell death. They achieve this critical effect by competitively binding to the peptidyl transferase center (PTC) located on the 60S ribosomal subunit, thereby effectively inhibiting the formation of peptide bonds, which is a core process in protein synthesis.
Looking ahead, the research space remains wide open for exploration, particularly given that almost all of the mechanistic studies conducted thus far have been carried out on the single fungus, *S. cerevisiae*. This highlights a significant opportunity for expanding investigations into a broader spectrum of fungal pathogens. Furthermore, ART899 the number of Amaryllidaceae alkaloids that have been thoroughly examined for their molecular actions is comparatively small when juxtaposed against the over five hundred known alkaloids identified from this rich plant family. These unexamined alkaloids, alongside other potential target compounds that can be accessed from ongoing phytochemical isolation efforts and synthetic studies, should form the foundational basis for uncovering even more detailed and novel mechanistic aspects of the antifungal effects exerted by Amaryllidaceae constituents.
Acknowledgements
The University of KwaZulu-Natal has provided significant and invaluable contributions towards the research endeavors undertaken by the participating authors, enabling the completion of this work.
Conflict of Interest
Both authors hereby jointly affirm and declare that there is no conflict of interest pertaining to any aspect of this undertaking or the publication of its findings.