Isocitrate lyase (ICL)[1]

In Candida albicans, Cyclo(L-Phe-L-Val) (32 μg/mL; 2 d) lowers the levels of icl product in MRC10 (Δicl), MRC11 (Δicl + ICL), and SC5314 (wild type), respectively [1].

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ICL Inhibitory Activity and Antifungal Activity of Diketopiperazines[1]
Isolated diketopiperazines were tested for ICL inhibitory activity according to methods reported previously. The inhibitory concentrations (IC50) values of the isolated compounds are shown in Table 1. Of the isolated diketopiperazines, cyclo(L-Pro-L-Leu) and cyclo(L-Pro-L-Val) demonstrated weak inhibitory activity toward the ICL enzyme, with IC50 values of 533.79 μM and 516.28 μM, respectively (Table 1). Cyclo(L-Phe-L-Val) exhibited the strongest inhibitory activity of the test compounds but demonstrated inhibitory potency that was less than that of 3-nitropropionate, with IC50 values of 109.50 μM and 15.95 μM, respectively (Figure 2a). The others exhibited no inhibitory activity. To determine the type of inhibition, kinetic analysis was performed with Cyclo(L-Phe-L-Val) (inhibitor) and phenylhydrazine (substrate). The inhibitor constant (Ki) was calculated from the Dixon plot. The results showed that Cyclo(L-Phe-L-Val) behaved as a mixed inhibitor, with a Ki value of 64.86 μM (Figure 2b). Fungal growth inhibition tests indicated that diketopiperazines at a concentration of 256 μg/mL did not exhibit inhibitory effects on SC5314 cultured in glucose (Table 1).

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Inhibition of C2 Substrate Utilization[1]
The glyoxylate cycle is necessary for virulence in C. albicans, which can survive in macrophages under abundant carbon sources, such as fatty acids or their breakdown products. When C. albicans is phagocytosed by a macrophage, the shift in metabolism from glycolysis to the glyoxylate cycle is activated so that the cells can utilize C2 carbon sources. It was expected that ICL inhibitors would reduce the nutrient uptake capacity and impede survival of the pathogen in the macrophage. To determine whether cyclo(L-Phe-L-Val) affects C2 substrate use, C. albicans strains SC5314, ATCC10231, ATCC10259, ATCC11006, and ATCC18804 were grown in YNB liquid broth containing either 2% glucose or 2% acetate as the sole carbon source. Cyclo(L-Phe-L-Val) exhibited a potent inhibitory effect on C. albicans in acetate (minimum inhibitory concentration of 32–64 μg/mL) but no inhibitory effect on C. albicans in glucose (Table 2). These results demonstrate that Cyclo(L-Phe-L-Val) affects ICL-mediated proliferation of the fungus under C2-carbon-utilizing conditions.

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Effects of Cyclo(L-Phe-L-Val) on Growth Phenotype and icl Expression[1]
To determine whether the cell phenotype of the icl-deletion mutant is affected by the presence of Cyclo(L-Phe-L-Val), a growth assay was conducted using C. albicans SC5314 (wild type) and two icl-deletion mutants (MRC10 and MRC11). After pre-culture, these strains were streaked onto YNB agar containing 2% glucose or 2% potassium acetate with or without 32 μg/mL Cyclo(L-Phe-L-Val). All strains grew normally on both the plates with glucose and with glucose plus the compound. However, MRC10 did not grow when acetate was the sole carbon source. Furthermore, none of the tested strains exhibited growth on the YNB agar plate with cyclo(L-Phe-L-Val) (Figure 3a). We further conducted semi-quantitative reverse-transcription (RT)-PCR to assess the effects of cyclo(L-Phe-L-Val) on ICL expression. No ICL-specific PCR product was detected in the cultures when SC5314 and MRC11 were grown in YNB liquid broth containing glucose. However, icl was strongly induced when these cells were cultured in YNB broth containing acetate. The intensity of the PCR band corresponding to the icl product decreased with increasing cyclo(L-Phe-L-Val) concentrations in the cells grown under icl expression conditions (Figure 3b). GPDH expression was detected in all treatments regardless of cyclo(L-Phe-L-Val) exposure. These results indicate that cyclo(L-Phe-L-Val) inhibits icl expression in C. albicans under C2-carbon-utilizing conditions.

ICL Inhibition Assay[1]
Before evaluation of the ICL inhibitory activity of the test compounds [Cyclo(L-Phe-L-Val)], the recombinant ICL from C. albicans was prepared according to methods described in a previous report. The ICL inhibitory activity of the test compounds was determined according to a previously described method. The ICL inhibition assay is based on the principle that glyoxylate phenylhydrazone forms in the reaction reagent after treatment with isocitrate and phenylhydrazine. Each test compound was dissolved in DMSO. The reaction mixture consisted of 20 mM sodium phosphate buffer (pH 7.0), 1.27 mM threo-DL(+)isocitrate, 3.75 mM MgCl2, 4.1 mM phenylhydrazine, and 2.5 μg/mL purified ICL and was incubated with or without the test compounds at a concentration range of 1–128 μg/mL at 37 °C for 30 min. The increase in intensity of absorbance resulting from the formation of glyoxylate phenylhydrazone was observed using a spectrophotometer at a wavelength of 324 nm. The inhibitory activity of the test compound was calculated relative to that of the DMSO control (n = 3). The methods for determination of the IC50 value and ICL enzyme concentrations have been described in previous reports. A known ICL inhibitor, 3-nitropropionate, was used as a reference control.

In Vitro Growth Assay[1]
C. albicans strains were cultured in YNB broth containing 2% glucose at 28 °C for 24 h, centrifuged at 15,000× g for 1 min, and washed twice with sterile distilled water. Each test compound [Cyclo(L-Phe-L-Val)] was dissolved in DMSO and diluted with YNB broth containing 2% glucose or 2% potassium acetate at concentrations ranging from 1 to 256 μg/mL. Additional DMSO was added to the medium at a final concentration of 0.5%. The fungal strain culture (20 μL) was poured into a 96-well assay plate at a final concentration of 1 × 104 cells/mL and total volume of 100 μL. The culture plates were incubated at 28 °C for 3 days. The positive control was amphotericin B, which is a known antifungal compound.

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Growth Phenotype and Icl Expression Analysis[1]
C. albicans SC5314 (wild type) and icl-deletion mutants (MRC10 and MRC11) were cultured in YNB broth containing 2% glucose at 28 °C on a rotary shaker for 24 h. Cells were collected and washed as described above. The harvested cells were streaked on YNB agar plates containing 2% glucose, 2% potassium acetate, or 2% potassium acetate plus 32 μg/mL Cyclo(L-Phe-L-Val) and incubated at 28 °C for 2 days. For icl expression analysis, the harvested cells were added to YNB liquid broth containing 2% glucose, 2% potassium acetate, or 2% potassium acetate plus Cyclo（L-Phe-L-Val） (8, 16, or 32 μg/mL) and incubated at 28 °C for 4 h. RNA extraction, cDNA synthesis, and semi-quantitative RT-PCR were performed using previously described methods. GPDH was used as the loading control.

[1]. Kim H, et al. Inhibitory Effects of Diketopiperazines from Marine-Derived Streptomyces puniceus on the Isocitrate Lyase of Candida albicans. Molecules. 2019 Jun 4;24(11):2111.

Cyclo-(L-Phe-L-Val) is an organooxygen compound and an organonitrogen compound. It is functionally related to an alpha-amino acid.
Cyclo-(L-Val-L-Phe) has been reported in Aspergillus sydowii with data available.

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The glyoxylate cycle is a sequence of anaplerotic reactions catalyzed by the key enzymes isocitrate lyase (ICL) and malate synthase, and plays an important role in the pathogenesis of microorganisms during infection. An icl-deletion mutant of Candida albicans exhibited reduced virulence in mice compared with the wild type. Five diketopiperazines, which are small and stable cyclic peptides, isolated from the marine-derived Streptomyces puniceus Act1085, were evaluated for their inhibitory effects on C. albicans ICL. The structures of these compounds were elucidated based on spectroscopic data and comparisons with previously reported data. Cyclo(L-Phe-L-Val) was identified as a potent ICL inhibitor, with a half maximal inhibitory concentration of 27 μg/mL. Based on the growth phenotype of the icl-deletion mutants and icl expression analyses, we demonstrated that Cyclo(L-Phe-L-Val) inhibits the gene transcription of ICL in C. albicans under C2-carbon-utilizing conditions.[1]

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Diketopiperazines possess various biological properties. Many research groups have focused on the pharmacological potential of diketopiperazines due to their advantages in medical chemistry, such as stability against proteolysis, mimicry of peptidic pharmacophoric groups, conformational rigidity, substituent group stereochemistry, and the existence of donor and acceptor groups for hydrogen bonding (which favor interactions with targets). Furthermore, diketopiperazines can be easily synthesized and isolated from natural products. In this study, five diketopiperazines, cyclo(L-Phe-L-Pro), cyclo(L-Pro-L-Leu), cyclo(L-Pro-L-Tyr), cyclo(L-Pro-L-Val), and Cyclo(L-Phe-L-Val), were obtained from a marine actinomycete, S. puniceus, using activity-guided separation processes. Among these diketopiperazines, cyclo(L-Pro-L-Leu), cyclo(L-Pro-L-Val), and cyclo(L-Phe-L-Val) exhibited ICL inhibitory activity, with IC50 values in the range of 27–112 μg/mL (Table 1). The structures of the isolated diketopiperazines exhibiting ICL inhibition activity contained an isopropyl moiety, which suggests that the isopropyl moiety plays a role in the inhibition of ICL activity.[1]

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In M. tuberculosis, a bacterium, and C. albicans, a fungus, virulence led to expression of icl, which encodes a component of the glyoxylate cycle during persistent infection of macrophages. These findings indicate that inhibitors of the glyoxylate cycle may reduce nutrient uptake capacity, leading to the death of these pathogens in macrophages. Thus, the effect of Cyclo(L-Phe-L-Val) on C. albicans ICL was investigated by analyzing the growth phenotype and icl transcript levels in C. albicans SC5314 (wild-type) and icl-deletion mutants (MRC10 and MRC11). The growth assay revealed that cyclo(L-Phe-L-Val) specifically inhibits the ICL enzyme, because no growth of SC5314 or MRC11 was observed on the YNB agar plates containing acetate plus 32 μg/mL of the compound. Moreover, icl transcript levels declined as a result of treatment with cyclo(L-Phe-L-Val). Overall, this research uncovered a compound of the diketopiperazine class as a promising antifungal agent that acts by suppressing C. albicans pathogenicity.[1]

C14H18N2O2

246.30

246.136827

35590-86-4

13783105

Typically exists as White to off-white solid at room temperature

1.525

2

2

3

18

322

2

CC(C)C1NC(=O)C(Cc2ccccc2)NC1=O

OQQPOHUVAQPSHJ-RYUDHWBXSA-N

InChI=1S/C14H18N2O2/c1-9(2)12-14(18)15-11(13(17)16-12)8-10-6-4-3-5-7-10/h3-7,9,11-12H,8H2,1-2H3,(H,15,18)(H,16,17)/t11-,12-/m0/s1

(3S,6S)-3-benzyl-6-propan-2-ylpiperazine-2,5-dione

cyclo(L-Phe-L-Val); 35590-86-4; Cyclo-(L-Val-L-Phe); (3S,6S)-3-benzyl-6-propan-2-ylpiperazine-2,5-dione; Cyclo-(L-Phe-L-Val); 3S-(1-methylethyl)-6S-(phenylmethyl)-2,5-piperazinedione; (3S,6S)-3-Benzyl-6-isopropylpiperazine-2,5-dione; (3S,6S)-3-benzyl-6-(propan-2-yl)piperazine-2,5-dione;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 8.33 mg/mL (33.82 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)