Constituents of Croton megalocarpus with Potential Anti-HIV Activity.

Reported herein is an anti-HIV monochlorinated compound, 1ß-acetoxy-3ß-chloro-5α,6α-dihydroxycrotocascarin L (1), of the rare crotofolane diterpenoid class. Compound 1, a suspected artifact of extraction, along with the previously undescribed 11ß-acetoxycrotocascarin L (2) and a known compound, crotocascarin K (3), were isolated from the bark of Croton megalocarpus, a Kenyan oil-producing seed crop. Compounds 1 and 3 inhibited HIV-1 replication with IC50 values of 28 and 5.5 nM, respectively. Furthermore, both compounds lacked cytotoxicity toward MT-4 cells and FM-55-M1 cells at concentrations of up to 50 µM. Compounds 1 and 3 were both found to inhibit HIV-1 protease.

Cytotoxic ent-abietane diterpenoids, banyangmbolides A-E, from the leaves of Suregada occidentalis.

The chemical investigation of a leaf extract from a herbarium specimen of Suregada occidentalis collected in Banyang Mbo Wildlife Sanctuary, Southwest Region, Cameroon, yielded five undescribed ent-abietane diterpenoids, banyangmbolides A-E, (1-5), and four known diterpenoids, gelomulides A (6), B (7), D (8) and O (9). The structures of the isolated compounds were determined using NMR, IR, ECD and HRESIMS. Compounds 5, 7 and 8, showed 48-55% inhibition at 200 µM against FM-55-M1 human melanoma cells.

Regional variation in the antibacterial activity of a wild plant, wild garlic (Allium ursinum L.).

Antibacterial activity is a common and highly studied property of plant secondary metabolites. Despite the extensive literature focusing on identifying novel antibacterial metabolites, little work has been undertaken to examine variation in levels of antibacterial activity in any plant species. Here, we used large-scale sampling of leaves of the antibacterial plant, wild garlic (Allium ursinum L.), assembling a set of tissue extracts from 168 plants, with 504 leaves collected and analysed. We assayed extracts for antibacterial activity against Bacillus subtilis and used LC-MS to carry out a chemometric analysis examining variation in individual metabolites, comparing them with several ecological parameters. We found that allicin was the only metabolite which was positively related to antibacterial activity. Soil temperature was a key determinant of variability in the concentrations of many foliar metabolites, however, neither allicin concentrations nor antibacterial activity was related to any of our measured ecological parameters, other than roadside proximity. We suggest that the synthesis of allicin precursors may be largely independent of growing conditions. This may be to ensure that allicin is synthesised rapidly and in sufficiently high concentrations to effectively prevent herbivory and pest damage. This finding contrasts with flavonoids which were found to vary greatly between plants and across sites. Our findings suggest that key biologically active metabolites are constrained in their concentration range compared to other compounds in the metabolome. This has important implications for the development of wild garlic as a health supplement or animal feed additive.

Fungal Drug Discovery for Chronic Disease: History, New Discoveries and New Approaches.

Fungal-derived drugs include some of the most important medicines ever discovered, and have proved pivotal in treating chronic diseases. Not only have they saved millions of lives, but they have in some cases changed perceptions of what is medically possible. However, now the low-hanging fruit have been discovered it has become much harder to make the kind of discoveries that have characterised past eras of fungal drug discovery. This may be about to change with new commercial players entering the market aiming to apply novel genomic tools to streamline the discovery process. This review examines the discovery history of approved fungal-derived drugs, and those currently in clinical trials for chronic diseases. For key molecules, we discuss their possible ecological functions in nature and how this relates to their use in human medicine. We show how the conservation of drug receptors between fungi and humans means that metabolites intended to inhibit competitor fungi often interact with human drug receptors, sometimes with unintended benefits. We also plot the distribution of drugs, antimicrobial compounds and psychoactive mushrooms onto a fungal tree and compare their distribution to those of all fungal metabolites. Finally, we examine the phenomenon of self-resistance and how this can be used to help predict metabolite mechanism of action and aid the drug discovery process.

Metagenomics Shines Light on the Evolution of "Sunscreen" Pigment Metabolism in the Teloschistales (Lichen-Forming Ascomycota).

Fungi produce a vast number of secondary metabolites that shape their interactions with other organisms and the environment. Characterizing the genes underpinning metabolite synthesis is therefore key to understanding fungal evolution and adaptation. Lichenized fungi represent almost one-third of Ascomycota diversity and boast impressive secondary metabolites repertoires. However, most lichen biosynthetic genes have not been linked to their metabolite products. Here we used metagenomic sequencing to survey gene families associated with production of anthraquinones, UV-protectant secondary metabolites present in various fungi, but especially abundant in a diverse order of lichens, the Teloschistales (class Lecanoromycetes, phylum Ascomycota). We successfully assembled 24 new, high-quality lichenized-fungal genomes de novo and combined them with publicly available Lecanoromycetes genomes from taxa with diverse secondary chemistry to produce a whole-genome tree. Secondary metabolite biosynthetic gene cluster (BGC) analysis showed that whilst lichen BGCs are numerous and highly dissimilar, core enzyme genes are generally conserved across taxa. This suggests metabolite diversification occurs via re-shuffling existing enzyme genes with novel accessory genes rather than BGC gains/losses or de novo gene evolution. We identified putative anthraquinone BGCs in our lichen dataset that appear homologous to anthraquinone clusters from non-lichenized fungi, suggesting these genes were present in the common ancestor of the subphylum Pezizomycotina. Finally, we identified unique transporter genes in Teloschistales anthraquinone BGCs that may explain why these metabolites are so abundant and ubiquitous in these lichens. Our results support the importance of metagenomics for understanding the secondary metabolism of non-model fungi such as lichens.

Disrupting iron homeostasis can potentiate colistin activity and overcome colistin resistance mechanisms in Gram-Negative Bacteria.

Acinetobacter baumannii is a Gram-negative priority pathogen that can readily overcome antibiotic treatment through a range of intrinsic and acquired resistance mechanisms. Treatment of carbapenem-resistant A. baumannii largely relies on the use of colistin in cases where other treatment options have been exhausted. However, the emergence of resistance against this last-line drug has significantly increased amongst clinical strains. In this study, we identify the phytochemical kaempferol as a potentiator of colistin activity. When administered singularly, kaempferol has no effect on growth but does impact biofilm formation. Nonetheless, co-administration of kaempferol with sub-inhibitory concentrations of colistin exposes bacteria to a metabolic Achilles heel, whereby kaempferol-induced dysregulation of iron homeostasis leads to bacterial killing. We demonstrate that this effect is due to the disruption of Fenton's reaction, and therefore to a lethal build-up of toxic reactive oxygen species in the cell. Furthermore, we show that this vulnerability can be exploited to overcome both intrinsic and acquired colistin resistance in clinical strains of A. baumannii and E. coli in vitro and in the Galleria mellonella model of infection. Overall, our findings provide a proof-of-principle demonstration that targeting iron homeostasis is a promising strategy for enhancing the efficacy of colistin and overcoming colistin-resistant infections.

Evidence for Natural Products as Alternative Wound-Healing Therapies.

Chronic, non-healing wounds represent a significant area of unmet medical need and are a growing problem for healthcare systems around the world. They affect the quality of life for patients and are an economic burden, being difficult and time consuming to treat. They are an escalating problem across the developed world due to the increasing incidence of diabetes and the higher prevalence of ageing populations. Effective treatment options are currently lacking, and in some cases chronic wounds can persist for years. Some traditional medicines are believed to contain bioactive small molecules that induce the healing of chronic wounds by reducing excessive inflammation, thereby allowing re-epithelisation to occur. Furthermore, many small molecules found in plants are known to have antibacterial properties and, although they lack the therapeutic selectivity of antibiotics, they are certainly capable of acting as topical antiseptics when applied to infected wounds. As these molecules act through mechanisms of action distinct from those of clinically used antibiotics, they are often active against antibiotic resistant bacteria. Although there are numerous studies highlighting the effects of naturally occurring small molecules in wound-healing assays in vitro, only evidence from well conducted clinical trials can allow these molecules or the remedies that contain them to progress to the clinic. With this in mind, we review wound-healing natural remedies that have entered clinical trials over a twenty-year period to the present. We examine the bioactive small molecules likely to be in involved and, where possible, their mechanisms of action.

A simplified and easy-to-use HIP HOP assay provides insights into chalcone antifungal mechanisms of action.

Elucidating the mechanism of action of an antifungal or cytotoxic compound is a time-consuming process. Yeast chemogenomic profiling provides a compelling solution to the problem but is experimentally complex. Here, we demonstrate the use of a highly simplified yeast chemical genetic assay comprising just 89 yeast deletion strains, each diagnostic for a specific mechanism of action. We use the assay to investigate the mechanism of action of two antifungal chalcone compounds, trans-chalcone and 4'-hydroxychalcone, and narrow down the mechanism to transcriptional stress. Crucially, the assay eliminates mechanisms of action such as topoisomerase I inhibition and membrane disruption that have been suggested for related chalcone compounds. We propose this simplified assay as a useful tool to rapidly identify common off-target mechanisms.

Multi-layered antimicrobial synergism of (E)-caryophyllene with minor compounds, tecleanatalensine B and normelicopine, from the leaves of Vepris gossweileri (I. Verd.) Mziray.

An aromatic alkaloid-rich 'absolute' extract from Vepris gossweileri inhibited Saccharomyces cerevisiae at 62.5 µg.mL-1 and Bacillus subtilis at 500 µg.mL-1. A loss of activity upon fractionation indicated possible synergistic effects. Three new acridones, gossweicridone A (3), B (4) and C (5) and known compounds from the extract were inactive. Combinations of compounds showed that a sub-fraction containing mixtures of minor compounds with (Ε)-caryophyllene augmented activity by 50-folds, with MIC values of 19.6 µg.mL-1 for S. cerevisiae and 375.0 µg.mL-1 for B. subtilis, demonstrating potent ΣFIC values of 0.02 and 0.375 respectively. From the active sub-fraction, three compounds were assigned as tecleanatalensine B, 13S-hydroxy-9Z,11E,15E-octadecatrienoic acid and normelicopine. In combination with (Ε)-caryophyllene they separately demonstrated MIC values of 18 µg.mL-1, 34 µg.mL-1 and 16 µg.mL-1, respectively against S. cerevisiae. The synergistic combinations were more potent with addition of pheophytin A, suggesting that the synergistic antifungal effect of the extract is multi-layered.

Ficus septica exudate, a traditional medicine used in Papua New Guinea for treating infected cutaneous ulcers: in vitro evaluation and clinical efficacy assessment by cluster randomised trial.

BACKGROUND AND OBJECTIVES: Infected cutaneous ulcers are major health problems for children living in rural areas of Papua New Guinea. The inaccessibility of affected populations and lack of access to basic healthcare, make a local plant-based therapy an attractive treatment option. We assessed Ficus septica exudate in biological assays relevant to wound healing. We then carried out a clinical trial to determine the exudate's efficacy in healing small cutaneous ulcers compared with Savlon antiseptic cream, and soap and water washing. METHODS: Pre-clinical in vitro assessment of the exudate was carried out using assays to monitor the pro-inflammatory responses of M1 macrophages and neutrophils, antibacterial assays using known ulcer pathogens, an Ames test for mutagenicity and LC-MS chemical analysis of the exudate. An open label cluster-randomised clinical trial was performed, enrolling participants from three different clusters with skin lesions less than 1 cm in diameter. Each cluster comprising 50 participants was randomly assigned to one of three treatment arms namely topical exudate, topical Savlon antiseptic cream, and standard care (soap and water treatment), all administered daily for 2 days. The primary outcome was clinical healing/improvement measured at days 7 and 14, assessed by three dermatologists using blinded photographs. The primary analysis was assessed as non-inferiority of F. septica treatment based on the risk difference for healing/improvement. RESULTS: In vitro, the exudate which is rich in the alkaloid ficuseptine, was found to be non-mutagenic whilst also inhibiting pro-inflammatory responses and exhibiting antibacterial activity. When administered to participants enrolled in the clinical trial, no significant differences were observed between the healing efficacy of F. septica exudate and the two comparator treatments (Savlon antiseptic cream and soap/water treatment). At day 14, but not at day 7, the efficacy of F. septica exudate for healing/improving the ulcers was non-inferior to Savlon antiseptic cream or water/soap treatment. CONCLUSIONS: F. septica exudate is non-mutagenic and has both bactericidal and anti-inflammatory properties. When applied topically to small cutaneous ulcers, the exudate has a healing effect that is non-inferior to Savlon antiseptic cream and standard treatment with soap and water at day 14. Our findings, which should be confirmed in larger clinical trials, have important public health implications.

Lepiniopsis ternatensis sap stimulates fibroblast proliferation and down regulates macrophage TNF-α secretion.

The sap of the tree Lepiniopsis ternatensis is used as a topical treatment for cutaneous leg ulcers in Papua New Guinea. This study, which is the first investigation of this medicinal plant, examines the effect of the sap on wound healing biology using human-derived primary cell lines. NMR spectra from 1D and 2D experiments revealed the sap to contain a single major component, identified as the polyphenol, trifucol. The sap significantly increased the proliferation of dermal fibroblasts at just 1.3 µg/ml, without influencing keratinocytes, suggesting a fibroblast-specific mechanism of stimulation. It also significantly inhibited TNF-α secretion by pro-inflammatory M1 macrophages, but not from neutrophils, at 130 µg/ml. The low toxicity of the sap towards dermal cells along with its fibroblast stimulation activity and downregulation of macrophage TNF-α makes it a potentially attractive agent to promote dermal wound healing in chronic non-healing ulcers.

Evaluation of Cypholophus macrocephalus sap as a treatment for infected cutaneous ulcers in Papua New Guinea.

Cypholophus macrocephalus sap is used to treat bacterially infected cutaneous leg ulcers in Papua New Guinea. High resolution LC-MS analysis of the sap revealed it to be rich in sulphated flavonoids. We assessed the effects of the sap on the differentiation and pro-inflammatory anti-microbial responses of M1 macrophages using IL-6 and TNF-α ELISAs and found significant increases in M1 macrophage IL-6 expression with concentrations as low as 243 ng/ml sap. Neutrophil IL-6 and TNF-α expression was also significantly increased but to a lesser degree. Matrix metalloproteinases (MMPs) 1, 2, 8 and 9 which are known to contribute to the toxic nature of wound exudates were inhibited by the sap at 24 µg/ml. The sap was tested with several bacterial species known to colonize cutaneous ulcers in Papua New Guinea but proved not to be active. Cypholophus sap stimulates pro-inflammatory, anti-microbial M1 macrophage and neutrophil responses at very low concentrations, whilst also inhibiting MMPs. The combination of an enhanced innate immune response and inhibition of MMPs in ulcer exudate, may contribute to the eradication of bacteria and healing of these infected ulcers. The sap concentrations used in these assays are readily achievable in an in vivo context.

Yeast Chemogenomic Profiling Reveals Iron Chelation To Be the Principle Cell Inhibitory Mode of Action of Gossypol.

Gossypol is an inhibitor of eukaryotic cells with an undetermined mode of action. Here we show that the chemogenomic profile of gossypol is strikingly similar to that of the iron chelators deferasirox and desferricoprogen. Iron import channels Fet1 and Fet3 are prominent in all three profiles. Furthermore, yeast inhibited by gossypol and deferasirox is rescued by the addition of Fe2+. We propose that Fe2+ chelation is in fact the principle mode of action of gossypol.

Lunacridine from Lunasia amara is a DNA intercalating topoisomerase II inhibitor.

An ethnobotanical survey of plants used to treat tropical ulcers in Papua New Guinea identified Lunasia amara as possessing anti-Staphylococcus aureus activity. Activity-guided fractionation of the aqueous bark extract resulted in the identification of the quinoline alkaloid lunacridine as the active principle. Lunacridine tends to cyslise at room temperature but the 2'-O-trifluoroacetyl derivative was found to be stable and therefore more suitable for biological assays. The compound exhibited a minimal inhibitory concentration (MIC) of 64 micro g/ml against Staphylococcus aureus NCTC 6571 and activity in the low micromolar range against HeLa and H226 cells; the latter showing signs of caspase-3/7 mediated apoptotic cell death. Experiments with drug resistant strains of Streptococcus pneumoniae suggested topoisomerase as a likely target for the drug in bacteria whilst decatenation assays with human topoisomerase II showed the compound to be a potent inhibitor of this isoform (IC(50)<5 micro M) thus explaining the drug's activity against human cell lines. Both lunacridine and 2'-O-trifluoroacetyl lunacridine exhibited mild DNA intercalation activity giving 50% decrease in ethidium DNA fluorescence at 0.22 and 0.6 mM, respectively, placing the drug amongst the DNA intercalating class of topoisomerase II inhibitors.

A Mini HIP HOP Assay Uncovers a Central Role for Copper and Zinc in the Antifungal Mode of Action of Allicin.

Garlic contains the organosulfur compound allicin which exhibits potent antifungal activity. Here we demonstrate the use of a highly simplified yeast chemical genetic screen to characterize its mode of action. By screening 24 validated yeast gene deletion "signature" strains for which hypersensitivity is characteristic for common antifungal modes of action, yeast lacking the high affinity Cu2+ transporter Ctr1 was found to be hypersensitive to allicin. Focusing on transition metal related genes identified two more hypersensitive strains lacking the Cu2+ and Zn2+ transcription factors Mac1 and Zap1. Hypersensitivity in these strains was reversed by the addition of Cu2+ and Zn2+ ions, respectively. The results suggest the antifungal activity of allicin is mediated through restricted Cu2+ and Zn2+ uptake or inhibition of Cu2+ and Zn2+ metalloproteins. As certain antimicrobial modes of action are much more common than others, the approach taken here provides a useful way to identify them early on.

Tropical ulcer plant treatments used by Papua New Guinea's Apsokok nomads.

ETHNOPHARMACOLOGICAL RELEVANCE: The tropical ulcer is a debilitating bacterial infection that is common in Papua New Guinea. Deploying healthcare infrastructure to remote and inaccessible rainforest locations is not practical, therefore local plants may be the best treatment option. Here we present an ethnobotanical survey of the tropical ulcer plant medicines used by the semi-nomadic Apsokok who roam the remote central mountains of Papua New Guinea's West New Britain Province. In vitro biological activity in assays relevant to tropical ulcer wound healing is also presented. MATERIALS AND METHODS: Focus groups and semi-structured interviews were used to acquire information on the uses of plants, vouchers of which were identified by comparison with authentic herbarium specimens. Antibacterial disc diffusion assays with Staphylococcus aureus and Fusobacterium ulcerans, MMP-9 enzyme inhibition and dermal fibroblast stimulation assays were carried out on plant saps and aqueous extracts of plant material. LC-MS was used to identify known plant metabolites. RESULTS: The ethnobotanical survey identified sixteen species that were used to treat tropical ulcers, all of which were applied topically. A subset of twelve species were investigated further in vitro. Four species produced zones of inhibition with S. aureus, all 12 species provided low level inhibition of MMP-9 and 8 species stimulated dermal fibroblast proliferation, although cytotoxicity occurred at higher concentrations. The extract of Homalium foetidum Benth. inhibited S. aureus and MMP-9 while at lower sub-cytotoxic concentrations stimulated fibroblast proliferation. Trans-3-O-p-coumaroylquinic acid cis-3-O-p-coumaroylquinic acid were detected in the aqueous extract of H. foetidum. CONCLUSIONS: Topical application of plant saps to wounds results in very high localised concentrations of plant metabolites which is likely to result in inhibition of MMP proteases. H. foetidum is a candidate plant for tropical ulcer treatment in remote areas.

Bridging the Gap to Non-toxic Fungal Control: Lupinus-Derived Blad-Containing Oligomer as a Novel Candidate to Combat Human Pathogenic Fungi.

The lack of antifungal drugs with novel modes of action reaching the clinic is a serious concern. Recently a novel antifungal protein referred to as Blad-containing oligomer (BCO) has received regulatory approval as an agricultural antifungal agent. Interestingly its spectrum of antifungal activity includes human pathogens such as Candida albicans, however, its mode of action has yet to be elucidated. Here we demonstrate that BCO exerts its antifungal activity through inhibition of metal ion homeostasis which results in apoptotic cell death in C. albicans. HIP HOP profiling in Saccharomyces cerevisiae using a panel of signature strains that are characteristic for common modes of action identified hypersensitivity in yeast lacking the iron-dependent transcription factor Aft1 suggesting restricted iron uptake as a mode of action. Furthermore, global transcriptome profiling in C. albicans also identified disruption of metal ion homeostasis as a potential mode of action. Experiments were carried out to assess the effect of divalent metal ions on the antifungal activity of BCO revealing that BCO activity is antagonized by metal ions such as Mn2+, Zn2+, and Fe2+. The transcriptome profile also implicated sterol synthesis as a possible secondary mode of action which was subsequently confirmed in sterol synthesis assays in C. albicans. Animal models for toxicity showed that BCO is generally well tolerated and presents a promising safety profile as a topical applied agent. Given its potent broad spectrum antifungal activity and novel multitarget mode of action, we propose BCO as a promising new antifungal agent for the topical treatment of fungal infections.

Medicinal plants of Papua New Guinea's Miu speaking population and a focus on their use of plant-slaked lime mixtures.

ETHNOPHARMACOLOGICAL RELEVANCE: Here we present the results of an ethnobotanical survey of the medicinal plants used by the Miu, a virtually unresearched ethnolinguistic group who live in the mountainous interior of Papua New Guinea's West New Britain Province. We compare the findings for those previously reported for the neighbouring inland Kaulong speaking population. Three species, Trema orientalis, Spondias dulcis and Ficus botryocarpa are used in combination with locally prepared slaked lime to produce intensely coloured mixtures which are applied to dermatological infections. Their effects on dermal fibroblast viability with and without slaked lime are examined. The sap of F. botryocarpa which is used to treat tropical ulcers was examined further with assays relevant to wound healing. MATERIALS AND METHODS: Focus groups and semi-structured interviews were used to acquire information on the uses of plants, vouchers of which were collected and identified by comparison with authentic herbarium specimens. LC-MS and NMR were used to identify chemical components. Cell viability assays were used to examine the effects of added slaked lime on dermal fibroblasts. For the sap of F. botryocarpa, fibroblast stimulation assays and antibacterial growth inhibition with Bacillus subtilis were carried out. RESULTS: The survey identified 33 plants and one fungal species, and clear differences with the inland Kaulong group despite their close proximity. Added slaked lime does not greatly increase the cytotoxicity of plant material towards dermal fibroblasts. The sap of F. botryocarpa contains the alkaloid ficuseptine as a single major component and displays antibacterial activity. CONCLUSIONS: The results demonstrate the potential for variation in medicinal plant use amongst Papua New Guinea's numerous language groups. The addition of slaked lime to plant material does not appear to present a concern for wound healing in the amounts used. The sap of F. botryocarpa displays antibacterial activity at concentrations that would occur at the wound surface and could be used as a highly accessible alternative to conventional antiseptics for remote communities in Papua New Guinea.

The haploinsufficiency profile of α-hederin suggests a caspofungin-like antifungal mode of action.

The leaves of common ivy (Hedera helix) contain the cytotoxic saponin α-hederin, which is inhibitory to Candida albicans at low concentrations. To investigate the mode of action of α-hederin, a haploinsufficiency screen was carried out using a library of 1152 Saccharomyces cerevisiae deletion strains. An ethanol ivy extract containing α-hederin was used in the initial screen to reduce the amount of compound required. Strains exhibiting disproportionately low growth were then examined in more detail by comparing growth curves in the presence and absence of α-hederin. This approach identified three hypersensitive strains carrying gene deletions for components of the transcription related proteins SWI/SNF, RNA polymerase II and the RSC complex. Saponin cytotoxicity is often attributed to membrane damage, however α-hederin did not induce hypersensitivity with an aminophospholipid translocase deletion strain that is frequently hypersensitive to membrane damaging agents. The haploinsufficiency profile of α-hederin is most similar to that reported for drugs such as caspofungin that inhibit synthesis of the fungal cell wall. Screening with plant extracts rather than isolated compounds, provides a valuable shortcut in haploinsufficiency screening provided hypersensitive strains are then confirmed as such using purified active principles.

A yeast chemical genetics approach identifies the compound 3,4,5-trimethoxybenzyl isothiocyanate as a calcineurin inhibitor.

The phosphatase enzyme calcineurin controls gene expression in a variety of biological contexts however few potent inhibitors are currently available. A screen of 360 plant extracts for inhibition of calcineurin-dependent gene expression in the model organism Saccharomyces cerevisiae identified the compound 3,4,5-trimethoxybenzyl isothiocyanate as an inhibitor. The compound was subsequently shown to inhibit human calcineurin via a mixed inhibition mechanism. To gain further mechanistic insight a yeast haploinsufficiency screen of 1152 deletion strains was carried out using a novel liquid medium screening method. The resulting haploinsufficiency profile is similar to that reported for the known calcineurin inhibitor FK506.