The present invention refers to new compounds deriving from spermatophyte plants of the Hypericaceae family, in particular from Hypericum mexicanum, that exhibit antifungal activity and relative pharmaceutical compositions and uses for the treatment of fungal infections.

Fungal infections are a health issue of increasing importance and the cause of mortality in immunosuppressed patients, subject to chemotherapy, prolonged antibiotic treatment or affected by HIV. Infections caused by Candida are particularly common, so much so that candidiasis is the most widespread fungal infection in the world and its medical treatment is often very difficult and expensive. A recent survey estimated the costs for antifungal therapies for a value of 8 billion/year in the United States alone (Global report WHO, 2014).

The drug classes currently in clinical use are azoles, echinocandins, polyenes and pyrimidines, substances which, in prolonged treatment have important side- effects such as renal and hepatic toxicity, fever, nausea and pharmacological counter-interactions (Zager, R.A. et al., 2000; Dismukes, W.E et al., 2000).

Furthermore, the effectiveness of clinical therapies is often threatened by the high development degree of resistance to antifungal agents such as azoles (i.e. fluconazole) on the part of many emerging pathogen fungal strains.

The search for alternative antiraycotic therapies and effective molecules is therefore of primary importance, as shown by the renewed interest of the pharmaceutical industry in the screening and isolation of new antimicrobial compounds of natural origin (Abreu A.N. et al., 2012).

Plants, in particular, have traditionally provided remedies for human health, and it is estimated that about 25% of the drugs used in a clinical setting are based on molecules of vegetable origin (Schmidt B. et al., 2008).

The Hypericum genus is a known source of therapeutic agents, including antidepressants, antimicrobial agents and anti-inflammatory principles. The use of Hypericum preparations is historically widespread in the traditional medicine of numerous countries.

The antifungal activity of essential oils and raw extracts has also been documented for many species of Hypericum (for example for H. perforatum, Tocci N. et al., 2013); for H. hyssopifolium and H. heterophyllum (Cakir A. et al., 2004); for H. caprifoliatum, H. carinatum, H. connatum, H. ternum, H. myrianthum, H. piriai and H. polyanthemum (Fenner R. et al., 2005); for H. linoides (Barros F.M.C. et al., 2013); for H. calycinum, (Decosterd L. et al., 1991); for H. patulum and H. mysorense (Mukherjee P.K. et al., 2002); for H. scabrum, H. scabroides and H. triquetrifolium (Kizil G. et al., 2004); for H. barbatum and H. rumeliacum (Saroglou V. et al., 2007); for H. havvae (Dulger G. and Dulger B., 2014); for H. riparium (Tala M.F. et al., 2013); for H. maculatum, Gudzic B. et al., 2002).

Despite this, the few studies available have attributed this activity to different classes of compounds, such as xanthones (Crockett S.L. et al. 2011), naphthodianthrones (Rezusta A. et al., 2012), chromenes (Decosterd L.A. et al., 1986) and flavanol glycosides (Yan-Hua L. et al., 2002).

A formulation containing a dimeric acylphloro-glucinol isolated from Hypericum gentianoides has proved to exert anti-HIV and anti-inflammatory properties (United States patent US 7,854,946).

Lipophilic extracts of the Hypericum species containing dimeric acylphloroglucinols have also shown antifungal activity against fungi of the genus Candida, Cryptococcus, Geothricum and Rodothorula (Barros F.M.C. et al., 2013), but no report of the direct antifungal activity of acylphloroglucinols is currently known.

The authors of the present invention have now found new compounds belonging to the group of dimeric acylphloroglucinols deriving from spermatophydc plants of the Hypericaceae family, in particular from Hypericum mexicanum, characterized by a high antifungal activity, reduced or even free of cytotoxicity, and efficacy against fluconazole-resistant pathogenic fungal strains,

Dimeric acylphloroglucinols isolated from H. mexicanum or other sources or obtained by synthesis, represent an effective alternative therapeutic instrument for the treatment of fungal infections in human beings and in animals, especially in the case of infections resistant to treatment with azoles. The compounds according to the invention are also characterized by practically zero cytotoxicity.

The authors have also developed a method for the isolation and purification of dimeric acylphloroglucinols of interest from plant tissues of Spermatophytes, in particular from the Hypericaceae family, preferably Hypericum mexicanum.

An object of the present invention therefore relates to dimeric acylphloroglucinol compounds having the following formula (I) uliginosine B-

(I)

(II) wherein Ri and R ₂ are each independently selected from linear or branched Ci-C ₄ alkyl groups _, optionally substituted with one or more halogen atoms, their relative enantiomers and/or derivatives or analogues thereof; provided that the groups Ri and R ₂ are not both methyl in formula (I).

The term derivatives according to the present invention refers to derivatives of esters or ethers at the level of each of the hydroxyl groups of both compounds having formula (I) and (II).

The term analogues, on the other hand, refers to classical isosteres, obtained by substituting one or more of the hydroxyls present on the meta-diphenol group with -SH or -NH ₂ groups. This definition also comprises the reduction products of the compounds indicated above that can be respectively obtained by means of:

- hydrogenation of the double bond on the saturated heterocycle with 6 carbon atoms of the compounds having formula (I) or

- dehydrogenation of the compounds having formula (II) to introduce one or more double bonds on the tetrahydropyran.

Further possible analogues relating to compounds having formula (I) alone provide for the introduction of one or more halogen atoms at the level of the double bond of the saturated heterocycle without destroying the pharmacophore (i.e. by reaction with lialogenated acids HCl, HBr, HI, or bromine or iodine, or also by acid catalyzed hydration reaction).

All of these derivatives or analogues of the compounds according to the invention can be prepared following the standard procedures of organic synthetic chemistry well-known to skilled persons in the field.

According to a preferred embodiment of the present invention, the compound having formula (II) is characterized in that the substituent groups Ri and R ₂ are both methyl.

According to a further preferred embodiment of the present invention, the compound having formula (I) or (II) is characterized in that the substituent groups Ri and R ₂ are both ethyl.

According to a yet another preferred embodiment of the present invention, the compound having formula (I) or (II) is characterized in that Ri is ethyl and R ₂ is methyl or vice versa.

These compounds can be isolated from plants belonging to the family Hypericaceae, preferably Hypericum, even more preferably from Hypericum mexicanum. The same compounds, however, can be obtained by traditional synthetic methods well-known to skilled persons in the field.

A further object of the present invention relates to a compound having formula (I) or (II) as defined above, for use in the human or veterinary medical field.

The invention also contemplates a pharmaceutical combination of at least two dimeric acrylphloroglucinol compounds, uliginosine B-like and sarothralen B-like as defined according to formulae (I) and (II), for use in the human or veterinary medical field. All the various combinations between compounds having formula (I) and (II), or "mixed" combinations of uliginosine B-like and sarothralen B-like compounds, or combinations of only uliginosine B-like compounds or only sarothralen B-like compounds, are possible. According to a preferred embodiment of the invention, the compounds having formula (I) and (II) or a combination of these compounds, are advantageously used for use in the treatment of systemic or local fungal infections.

For illustrative and non-limiting purposes, the fungal infections that can be treated by means of the compounds of the present invention are selected from the group consisting of Candidiasis (i.e. vaginal, systemic, urinary, esophageal, oral, subungualeal Candidiasis), Cryptococcosis, Coccidioidomycosis, Histoplasmosis and Sporotrichosis.

A further object of the present invention relates to a pharmaceutical composition comprising a compound having formula (I) and (II) or a combination of these compounds as active principle, together with one or more pharmaceutically acceptable adjuvants and/or excipients.

The pharmaceutical compositions according to the invention can be administered orally, parenterally, transdermally, rectally, nasally, buccally, topically or by controlled-release implant.

The pharmaceutical compositions according to the invention can therefore be in the form of tablets, suppositories, pills, capsules, suspension, solution, granules, plugs, vaginal tablets, gynecological cream, ointment, varnish, lotion, skin spray, powder or ovules.

The quantity of active compound to be administered depends on the particular pathology the subject to be treated is suffering from, the weight and age of the subject, the administration method selected and the doctor's opinion.

The treatment scheme provides for the administration of the drug in dosages ranging from 0.0001 to 10 mg/kg/day.

According to an exemplificative but non-limiting embodiment of the present invention, said pharmaceutical composition suitable for oral administration (i.e. in the form of tablets or pills) comprises a compound having formula (I) or (II) or a combination thereof, in a dosage selected from 50, 100, 200, 400, 600, or 800 mg. According to a further exemplificative but non-limiting embodiment of the present invention, said pharmaceutical composition suitable for topical administration (i.e. in the form of a gel, cream, lotion or varnish) comprises a compound having formula (I) or (II) or a combination thereof, in a weight percentage ranging from 0.05% to 5%, preferably from 0.5% to 1% (corresponding to a concentration range of 1 mM-100 mM, preferably 10 mM-20 mM) with respect to the total weight of the composition.

The composition also relates to the use of the pharmaceutical compositions according to the invention, for the treatment of systemic or local fungal infections. Finally, the invention contemplates a method for isolating the compounds having formula (I) and (II) from a plant of the Hypericum genus, comprising the following steps:

a) obtaining an extract of dried leaves or stems of the plant Hypericum with hexane;

b) extraction of the product of step a) with a mixture of acetonitrile in acidic water (70:30);

c) separation of the fraction obtained in step b) and gradient elution by chromatography using a solvent comprising methanol and 0.5% formic acid;

d) isolation of the compounds from the fraction separated in step c) by means of high-performance liquid chromatography with a mobile phase consisting of a first solvent comprising methanohwater 7:3 v/v with 0.5% formic acid, and a second solvent comprising methanol and 0.5% formic acid.

Said plant preferably belongs to the Hypericum mexicanum species.

The present invention is now described, for illustrative but non-limiting purposes, according to a preferred embodiment with particular reference to the attached figures, in which:

- Figure 1 shows the growth curve of a Candida albicans species, non-treated and treated with the hexane extract of Hypericum mexicanum.

- Figure 2 shows the chromatogram (A) and UV spectrum (B) of fraction I.

- Figure 3 shows the chromatogram and UV spectra of fraction III- IV.

- Figure 4 shows the evaluation of the cytotoxicity of fraction III-IV on human cell lines.

- Figure 5 shows the structures of uliginosine B-like compounds 1, 2 and 3.

- Figure 6 shows the structures of sarothralen B-like compounds 4, 5 and 6.

- Figure 7 shows a histogram illustrating the evaluation of the cytotoxicity of dimeric acylphloroglucinols on human cell lines.

- Figure 8 shows the MS spectra of uliginosine B-like compounds 1, 2 and 3.

- Figure 9 shows the MS spectra of sarothralen B-like compounds 4, 5 and 6.

- Figure 10 shows the results of the chemogenomic screening relating to compound 4 having formula II.

The following examples are provided for illustrative purposes only of the present invention and should not be considered as limiting the protection scope, as defined by the enclosed claims.

EXAMPLES

EXAMPLE 1 : Isolation and characterization of antifungal activity of compounds extracted from H. mexicanum

The dried leaves and stems of the plant Hypericum mexicanum were subjected to three consecutive extractions by grinding with hexane.

The extract obtained was tested against pathogenic fungi in accordance with the protocol of the European Antimicrobial Sensitivity Testing Committee (EUCAST Definitive Document EDEF 7.2 2012 revision).

The inhibitory properties were expressed as the minimum inhibitory concentration (MIC), corresponding to the quantity of extract necessary for inhibiting 50% of the growth of the fungi.

Before the test, the extract was dissolved in DMSO. The concentration of DMSO used (1%) did not affect the growth of the fungi.

The extract was able to strongly inhibit the growth of pathogenic fungi of the genus

Candida as shown in Table 1 hereunder.

Table 1 : Antifungal activity of the extract of H. mexicanum

GM: geometric mean

The inhibition proved to depend on the concentration (see Figure 1).

The data are expressed as geometric mean of the MIC values (mean + standard deviation) of at least 3 replicates for each strain of Candida spp.

The extract was further purified through liquid/liquid extraction with a mixture of acetonitrile in acid water (70:30) to remove the inactive components. The remaining fraction in hexane was tested for the antifungal activity according to the procedure described above and the activity is shown in Table 2 hereunder. Table 2: Antifungal activity of fractions isolated from the extract of Hypericum mexicanum

*GM: geometric mean

The final fraction referred to in the previous point was subjected to preparative column chromatography using Si0 ₂ as stationary phase and a hexane/ethyl acetate mixture as mobile phase, in an elution gradient (stalling with 100% hexane and up to 1: 1 of hexane/ethyl acetate). Various fractions were collected and subjected to thin-layer chromatography (TLC silica gel 60 T254, Merck, Germany), using hexane/ethyl acetate 9: 1 as mobile phase.

The analytes were visualized by carbonization with an aqueous solution at 10% of cerium (IV) sulfate and 15% H ₂ S0 ₄ .

The fractions attributable to the same compound were joined, and the pools obtained were subjected to further analyses, antimycotic tests, and characterized by means of NMR and LC-MS spectrometry.

The LC-MS measurements were effected using a Shimadzu High Performance LC (CBM-20 A system, with a LC-20AB binary pump, Italy) with a Kinetex C18 column (pore size 100 A, 4.6 mm ID, particle size 2.6 microns, and a length of 10 cm, Phenomenex, Italy).

The mobile phase was composed of solvent A, methanol: water (7:3 v/v) with formic acid 0.5%, and solvent B, methanol with formic acid 0.5%. The elution gradient program was adapted to each of the fractions tested:

1. Fraction I: isocratic elution, 70% B and 30% A, flow-rate: 0.9 ml/min;

2. Fractions III-IV: gradient elution, starting with 75% B (92.5% MeOH, 7.5% H ₂ 0) and reaching 85% B (95.5% MeOH, 4.5% H ₂ 0) in 20 minutes. The flow- rate was set at 1.1 ml/min. The eluate was simultaneously subjected to spectrophotometric analysis with a UV/VIS spectrometer and mass spectrometry analysis with a triple quadrupole mass spectrometer API 3000 (Applied Biosystems) equipped with an electrospray ion source (ESI). Both the positive and negative ion scans were acquired, and the mass spectrometer was used in full scan mode, and also in scan mode of the ions produced, in order to construct fragmentation models used for the elucidation of the structure.

From the analyses, Fraction I proved to be mainly composed of a single species (see Figure 2), whereas Fraction III-IV proved to be composed of six compounds (see Figure 3).

The fractions were tested for antifungal activity, as previously described, and Fraction III-IV showed a strong antimycotic activity, whereas no activity was detected for Fraction I, as shown in Table 3 hereunder.

Table 3.

Table 3: Antifungal activity of the hexane extract of H. mexicanum

GM: geometric mean In order to evaluate the specific activity of the fraction III-IV for pathogenic fungi, a cytotoxicity test of the same was carried out against human cell lines: Hs27 (ATCC® CRL-1634™) (fibroblasts) and peripheral blood mononuclear cells (PBMC).

The cytotoxicity was tested using the WST-8 assay (4-[3-(2-methoxy-4- nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolium]-l,3-benzene sodium disuifonated salt). The cells were seeded in 96-well culture plates at a concentration of 5,000 cells/well and incubated for 24 hours before treatment. Before the test, the fraction III-IV was diluted in DMSO and added at a final concentration of 64.32 and 16 μg/ml (final volume of DMSO 0.5% v/v).

In order to measure the cytotoxic effects, 10 μΐ of solution of the Kit-8 (Sigma- Aldrich) (containing the solution WST-8) were added to each well and left to incubate for 2 hours and 4 hours for fibroblasts and PBMC, respectively, at 37°C before reading the absorbance at 450 nm. The results were analyzed with the Student t-test.

The data are expressed as percentage of vitality and standard error of the mean. No significant cytotoxic effect was observed for the fraction III-IV (see Figure 4), showing that the same has a growth inhibition specificity of fungal cells but not towards human ceils.

The final, isolation of the compounds present in fraction III-IV (Figure 3) was carried out through high-performance chromatography on a Waters Alliance 2695 instrument, equipped with a DAD detector and Kinetex C18 column (pore size 100 A, ID 4.6 millimetres, particle size 2.6 microns, and length 10 cm, Phenomenex, Italy) with a mobile phase composed of solvent A, methanohwater (7:3 v/v) with formic acid 0.5%, and solvent B, methanol with formic acid 0.5%. The elution was effected in gradient starting with 70% B and reaching 100% B in 26 minutes.

The analytes isolated easily ionize in negative ion mode; Table 4 hereunder summarizes the main ions observed for each of the six molecules.

Table 4: Main ions (negative ion mode) observed in the HPLC-MS analysis in full scan and ion scan

The structure of the compounds isolated was characterized through NMR spectroscopy with a Bruker Avance-400 MHz spectrometer, operating with a stationary magnetic field having a force of 9.4 T and equipped with a 5 mm BBI probe. The proton 90° pulse length was calibrated at 9.4 ms, with a transmission power of 0 dB. The temperature was kept constant at 300.2 K. The residual proton solvent was used as internal reference for the chemical shift scale (CHCI3, 51H: 7.26 ppm, 5 ¹³ C: 77.16 ppm).

The NMR assignments allowed compounds 1, 2 to be classified as B-like uliginosine compounds and compounds 4, 5 as sarothralen B-like compounds. The structures of compounds 3 and 6 were elucidated only on the basis of MS and MS/MS full scan measurements as they were isolated in small quantities. 1. 1H-NMR resonances of compound 1 [ppm]: 6.70 (d, 9.9 Hz), 5.44 (d, 9.9 Hz), 4.21 (sept, 6.7 Hz), 3.89 (sept, 6.7 Hz), 3.54 (br m), 1.53 (br s), 1.48 (br s), 1.45 (br s), 1.18 (br s)

2. ¹ H-NMR resonances of compound 2 [ppm]: 6.70 (d, 9.9 Hz), 5.44 (d, 9.9 Hz), 4.21 (sept, 6.7 Hz), 3.81 (sext, 6.7 Hz), 3.54 (br m), 1.88 (m), 1.53 (br s), 1.48 (br s), 1.45 (br s), 1.42 (m), 1.18 (d, 6.7 Hz), 0.94 (t,7.5 Hz)

3. ¹³ C-NMR resonances of compound 2 [ppm]: 210.9, 210.8, 200.2, 199.4, 171.7, 162.2, 159.3, 155.4, 124.6, 117.3, 111.2, 108.1, 107.1, 104.2, 103.6, 78.1, 45.7, 44.8, 36.6, 28.1, 27.7, 26.4, 24.9, 19.3, 19.1, 16.9, 16.7, 11.9

4. ¹ H-NMR resonances of compound 4 [ppm]: 5.19 (t, 6.9 Hz), 4.21 (sept, 6.9 Hz), 3.89 (sept, 7.0 Hz), 3.54 (m), 2.83 (dt, 17 Hz, 5.3 Hz) , 2.25 (m), 2.15 (m),

1.82 (br s), 1.72 (br s), 1.61 (br s), 1.47 (br s), 1.17 (d, 6.9 Hz)

5. ¹³ C-NMR resonances of compound 4 [ppm]: 210.7, 206.9, 203.3, 199.4, 187.3, 171.6, 161.6, 161.4, 133.1, 122.1, 111.3, 107.1, 105.2, 103.6, 102.4, 79.5, 44.2, 40.7, 38.9, 36.5, 30.8, 29.4, 27.5, 25.8, 22.7, 22.1, 19.5, 19.2, 17.8, 16.9, 16.0

6. ¹ H-NMR resonances of compound 5 [ppm]: 5.19 (t, 6.9 Hz), 4.21 (sep, 6.9 Hz), 4.12 (sext, 7.4Hz), 3.54 (m), 2.83 (dt, 17 Hz, 5.3 Hz), 2.25 (br), 2.15 (br), 1.82 (br), 1.72 (s), 1,61 (s), 1.47 (s), 1.27 (br), 1.24 (d, 7.2 Hz), 1.17 (d, 6.9 Hz ), 0.89 (t, 6.9 Hz).

The structures of compounds 1, 2 and 3 are illustrated in Figure 5, whereas Figure 6 shows the structures of compounds 4, 5 and 6 obtained by combining the information obtained by means of MS and NMR analyses.

Compound 1 having formula (I) wherein Ri and R ₂ are both methyl was already known in literature (Meirelles G. et al., 2012; Barros F.M.C. et al. 2013) as compound uliginosine B (CAS-no. 19809-79-1) having an antifungal activity and coming from plants of the genus Hypericum. The spectral characteristics of the compounds isolated were registered on a Hitachi U-2000 UV/VIS spectrophotometer using quartz cuvettes with a 10 mm optical path.

For the determination of the molar absorption coefficient (ε), the compounds were dissolved in hexane. For each compound, two solutions were prepared at a concentration of 10 and 20 mg/1, respectively.

The experimental ε coefficients of the new isolated compounds were as follows: Compound 1: 19195 (280 nm)

Compound 2: 19592 (280 nm)

Compound 3: 3909 (280 nm)

Compound 4: 16375 (280 nm)

Compound 5: 15045 (280 nm)

Compound 6: 7196 (280 nm)

The high-resolution MS spectra were recorded with HDMS-QTOF Synapt (Waters Corp., Milford, MA). The HDMS analysis was performed in ESI using leucine encephalin as the MS calibrating solution (see Figures 8 and 9).

EXAMPLE 2: Antifungal activity test of dimeric acyiphiorogiucinols isolated from Hypericum mexicanum

The dimeric acyiphiorogiucinols isolated from Hypericum mexicanum were tested individually for their antifungal activity following the procedures previously described.

All the compounds isolated showed a strong antifungal activity with respect to Candida spp at concentrations ranging from 4 to 32 μg/ml, as shown in Table 5 hereunder.

Table 5: Antifungal activity of dimeric acyiphiorogiucinols isolated from Hypericum, mexicanum Species GM* MICso (μίξ/ηιΐ)

(nr. strains)

Compound

2 3 4 5 6

C. albicans (2) 16 8 8 4 4 4

C. glabrata (2) 32 16 16 8 4 4

GM: geometric mean

** 1 : Known compound, Uliginosine B

All the compounds 2-6 are more effective than the known compound used as reference, Uliginosine B already known in literature.

As an example of the potential of use of dimeric acylphloroglucinols in the treatment of fungal infections, compounds 2 and 5 were tested against a wide range of clinical isolated fungal strains, both sensitive and resistant to azoles, and the activity was compared to that of fluconazole, the main antifungal agent used in clinical practices.

The antifungal test was carried out as previously described in Example 1. The dimeric acylphloroglucinols showed a powerful antifungal activity against all the fungi tested and in particular against pathogens resistant to azoles with MIC50 values ranging from 8 to 30 mM. The results are shown in Table 6 hereunder.

Table 6: Antifungal activity of dimeric acylphloro-glucinols isolati da H. mexicanum

Species GM* MICso (μΜ)

(nr. strains)

Compound Compound Fluconazole

2 5

Candida albicans (4) 12.52 8 61.5

Candida parapsilosis 32 16.73 65.52 (3)

Candida lusitaniae 16 30 1.16

(2)

Candida pararugosa 8 15 0.4

(1)

GM: geometric mean

The cytotoxicity of compound 2 and compound 5 was tested against Hs27 fibroblasts (ATCC® CRL-1634™) and peripheral blood mononuclear cells (PBMC) following the procedure previously described. The concentration of the compound inhibiting 50% of the cell growth (IC50) of the compounds, proved to be higher than 65 μΜ, therefore no relevant cytotoxicity was observed at active concentrations against fungal pathogens, confirming the specific antimycotic activity of the molecules (see Figure 7).

EXAMPLE 3: Study on the mechanism of action of the dimeric acylphloroglucinols according to the invention

Compound 4 having formula (II) was selected and subjected to a chemogenomic screen in order to identify possible molecular targets.

Pools of diploid yeast strains were used, each of which carries a specific deletion

(Yeast Deletion Heterozygous Diploid Pools - Invitrogen Cat. no.95401.H4Pool).

The method used is similar to that described in detail in the publication of

Robinson D. G. et al. 2014.

The pool of 5936 yeast mutants was grown for 20 generations in the presence of compound 4 having formula (II) (150 and the control (1% DMSO). The Log2 ratio (intensity of the control/intensity of the samples treated) was calculated and graphically represented in relation to the single genes.

Fieure 10 shows the chemogenomic screen relating to compound 4 having formula (II),

This chemogenomic screen revealed deletion strains highly sensitive to the drug tested. The most sensitive mutant was heterozygous for chaperonin CCT5, a subunit of the cytosolic chaperonin containing TCP-1 (CCT) required for the assembly of actin and tubulins (points identified by the abbreviations in the chart; logFC >1.5 and P <0.01).

CCT assists the folding of various proteins that are important in cell cycle regulation, cytoskeletal assembly and chromatin remodelling (Dekker C. et al. 2011). It also assists the correct assembly and functioning of the septin ring (Dekker C. et al. 2008).

Even more significantly, the interactions between CCT and its major substrates actin and tubulin appear to be sequence- specific (Brackley K.I. and Grantham, 2009) which would make it an extremely important pharmaceutical target, especially due to the evidence that changes in expression of a subunit of the CCT can block hyphal morphogenesis in Candida (Rademacher F. et al., 1998).

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