Tacrolimus as Antifungal Agent

Tacrolimus (FK506) is an immunosuppressant drug widely used to avoid organ rejection in transplant patients. It has a profound influence on the cellular stress response by interfering with the calmodulin-calcineurin signaling pathway. In this context FK506 also became a valuable antifungal drug in medical care. Here it is shown in vitro that tacrolimus has a potent growth inhibition activity against 11 fungi and 3 oomycetes of agricultural importance. The significance of this finding is discussed with respect to crop protection. The in silico molecular docking to 6 major antifungal enzymes determined UDP-N-acetylmuramoyl-L-alanine: D-glutamate ligase (MurD) as the main target by the best affinity score.


Introduction
Presently azoles, echinocandines, pyrimidine analogs and polyenes are among the most common antifungals in human medical care and crop protection.The search for new potent antifungal agents will remain an urgent task at the background of an ongoing emergence of drug resistance. 1 Especially resistant pathogenic fungi rise concerns human medical care.For instance, immunocompromised patients after HIV infection or organ transplantation, who have overcome cryptococcal meningitis infection, may need a life-time fluconazole prophylaxis. 2Here, management of infections become extremely difficult if azole resistant pathogens get involved.Also the arise of fluconazole-resistant mutants among Cryptococcus neoformans clinical isolates was observed. 3In search for new targets for antifungals the calmodulin-calcineurin signaling cascade came into focus.A stress response of a fungal cell starts by the uptake of Ca 2+ , which then binds to the four binding sites of calmodulin.After a conformational change Ca 2+ occupied calmodulin forms a ternary complex together with calcineurin subunits CnA and CnB, thereby gaining a phosphatase activity; in turn the complex dephosphorylates transcription factor Crz1. Genes activated by dephosphorylized Crz1 are involved in drug resistance, growth and cell wall integrity.Generally, calcineurin is an important part of the stress response of fungal cells. 4earching for novel immunosuppressive agents a 822-kD 23-membered macrolide lactone named tacrolim- us (FK506, Fujimycin, etc.) was isolated and characterized from Streptomyces tsukubaensis (Fig. 1). 5 Tacrolimus in capsules for oral intake and as solution for injection is used for prophylaxis against organ rejection in patients receiving liver, kidney or heart transplants. 6As a topical ointment it is applied as a second-line therapy for the short-term and non-continuous chronic treatment of moderate to severe atopic dermatitis in non-immunocompromised adults and children.FK506 has a favorable toxicological profile: no evidence of mutagenicity was observed in vitro in the CHO/HGPRT assay (the Chinese hamster ovary cell assay).Also it did not cause unsched-Antypenko et al.: Tacrolimus as Antifungal Agent uled DNA synthesis in rodent hepatocytes.LD 50 in rat oral test was 134 mg/kg for tacrolimus hydrate. 7xperimental evidence suggests that tacrolimus binds to the intracellular protein FKBP12 and thereby interferes with the calcineurin signal pathway, which is a key element in regulation of intracellular Ca 2+ concentration. 4FKBP12-tacrolimus inhibits the phosphatase activity of the calmodulin-CnA-CnB complex, which in turn silences transcription factor Crz1 and corresponding stress related genes.As a consequence, inhibitors of calcineurin like tacrolimus may function as potent antifungals.In combination with standard antifungal drugs FK506 may reverse resistance against them or increase their efficacy. 8,9Also phytopathogenic fungi rely on the calcineurin pathway securing cell wall and membrane integrity, virulence 10 or formation of infectious appressoria. 11ractically all of tacrolimus studies are related to the development of supportive antifungal therapy to improve regimes of infected patients.Here we describe novel antifungal efficacy of FK506 towards eleven pathogenic fungi and three Phytophthora oomycetes of agricultural importance.

1. Known Antifungal Data
All reported antifungal literature data were summarized below (Table 1) to show its found minimum inhibitory concentrations (MIC).
Lowering the concentration to 0.05 µg/mL still conferred an inhibition of all strains.A. alternata, F. graminearum, B. cinerea, C. higginsianum and A. niger were the most sensitive ones and were effected more than 70%.

3. Molecular Docking Studies
In order to propose unstudied antifungal activity mechanism in silico molecular docking was done 34 .The found affinity scores of tacrolimus and reference cyproconazole 33 to common antifungal targets 35 are presented in Table 3.
As it was expected the 14α-demethylase (CYP51) has shown the best affinity with triazole derivative cyproconazole according to its best score (-7.9 Kcal/Mol) (Table 3).
An interesting result was found for tacrolimus, because it fitted into the active sites of secreted aspartic proteinase (SAP2) and UDP-N-acetylmuramoyl-L-alanine: D-glutamate ligase (MurD) better then cyproconazole, and its affinity scores were much higher (-8.5 and -9.8 Kcal/Mol correspondingly).
The visualization of interaction (Figure 2) with the best target indicates that tacrolimus binds to MurD by four conventional hydrogen and three carbon hydrogen bonds.Also five hydrophobic alkyl and π-alkyl bonds are formed with LEU15, LEU416, ARG186 and PHE422 residues of enzyme (Table 4).

Experimental
The mycelial growth rate assay was used for antifungal studies. 36Strains of filamentous fungi were obtained from the following sources: Aspergillus niger DSM 246, Altenaria alternata DSM 1102, Fusarium equiseti DSM 21725, F. graminearum DSM 1095, F. fujikuroi DSM 893, Verticillium lecanii, Penicillium digitatum DSM 2731 from DSMZ (Braunschweig, Germany); Fusarium oxysporum 39/1201 St. 9336 and Botrytis cinerea from the Technische Universität Berlin (Germany); Colletotrichum higginsianum MAFF 305635, originally isolated in Japan, via the Department of Biology, Friedrich-Alexander-Universität (Erlangen, Germany); Phytophthora infestans GL-1 01/14 wild strain, p-3 (4/91; R+) and p-4 (4/91; R-) strains were kindly donated by Julius Kühn-Institut (Quedlinburg, Germany).Potato Dextrose Agar (PDA) were purchased from C. Roth (Karlsruhe, Germany).Cyproconazole (99%) was obtained from (Sigma Aldrich, Germany).Tacrolimus (99 %) was purchased from Huaian Ruanke Trade, Ltd. (Huaian, China).Strains were cultivated on PDA for 6 d at 25 °C.Spores from each strain were gently harvested with a sterile glass rod from plate surfaces with deionized water.Spore concentration numbers in suspension were determined microscopically and adjusted to 7.5*10 6 spores/mL.The clear stock solutions of 1 mg/mL were made of 0.01 g of cyproconazole and tacrolimus in 10 mL of deionized sterile water as solvent.10 mL of these stock solutions were mixed in situ into 99 mL of PDA prior to solidification to obtain a final concentration of 1 µg/mL.Additionally, from the same stock solution PDA solutions with final concentration of 0.05 and 0.001 µg/mL were made appropriately.9 mL of each mixture were poured

#
Protein preparation.Discovery Studio 4.0 was used to delete water molecules and ligand from crystal structures.The proteins were saved as pdb-files.In AutoDock-Tools-1.

Conclusion
Considering a substantial role of FK506 in crop protection due to its distinct activity against phytopathogenic fungi, the availability in bulk quantities at affordable costs have to be taken into account. 38For the development of a formulation for agricultural usage purity standards may be much less demanding than for immunosuppressant manufacturing.Furthermore, quantities necessary of tacrolimus may be reduced if advantages of synergistic effects in blends with conventional antifungal agrochemicals (e.g.triazoles) can be generated as it is already described for medical care of patients infected with C. albicans. 8Investi-gations to further explore the potential benefits of FK506 in agriculture must be accompanied by studies covering emergence of resistance, toxicity and environmental friendliness of this compound.

Table 4 .
The list of in silico calculated bonds formed between tacrolimus and UDP-N-acetylmuramoyl-L-alanine: D-glutamate ligase (MurD) active site

Figure 2 .
Figure 2. Visual representation (2D (left) and 3D (right)) of the tacrolimus showing bonds formation and position in the active site of UDP-N-acetylmuramoyl-L-alanine: D-glutamate ligase (MurD) 1UAG.Pale green -van der Waals interaction, green -classical conventional hydrogen bond, light green -non classical carbon hydrogen bond, pink -hydrophobic alkyl and π-alkyl bonds.

Table 1 .
Summary of reported antifungal activity of tacrolimus (FK506)