iKIX1 targets the interaction between the transcription co-activator Mediator complex and the pleiotropic drug resistance (CgPdr1) transcription factor in Candida glabrata. Specifically, it inhibits the interaction between the KIX domain of the mediator subunit CgGal11A and the activation domain of CgPdr1. By blocking this interaction, iKIX1 inhibits Pdr1-dependent gene activation and resensitizes drug-resistant C. glabrata to azole antifungals.

In the presence of 5 µM ketoconazole (KET), iKIX1 (10–20 μg/ml) reduces cell proliferation in HepG2 cells in a concentration-dependent manner [1]. With a fitted Kd of 319.7 nM, the FP titration curve illustrates the interaction between the CgGal11A KIX domain and the CgPdr1 AD30. CgPdr1 AD30 and iKIX1 are in competition with an IC50 of 190.2 µM. During in vitro binding experiments, iKIX1 demonstrated an apparent Ki of 18 µM and a Kd of 0.32 µM for the CgPdr1 activation domain (AD) of the CgGal11A KIX domain [1]. In the Sc pdr1Δpdr3Δ strain, which carries plasmid-borne CgPDR1 and 3XPDRE-luciferase, iKIX1 (0-50 µM) dose-responsively suppresses the increase of luciferase activity produced by KET [1]. In Saccharomyces cerevisiae, the recruitment of Gal11/Med15 to Pdr1-regulated target genes was investigated using the chromatin immunoprecipitation (ChIP) assay. Gal11/Med15 is quickly recruited by ketoconazole to the promoters of Pdr1 target genes PDR5 and SNQ2. Ketoconazole-induced Gal11/Med15 recruitment is eliminated by iKIX1, and azole-induced ScPdr1 target gene transcription is significantly suppressed [1]. There is an impact of iKIX1 (20 μM) on C's transcription. glabrata Pdr1-regulated genes involved in drug efflux and MDR (CgCDR1, CgCDR2, and CgYOR1). iKIX1 by alone had no discernible impact on Pdr1 target gene induction. On the other hand, iKIX1 pretreatment permanently and concentration-dependently decreased ketoconazole-induced CgPdr1 upregulation [1]. C was the subject of an RNA sequencing (RNA-Seq) experiment. glabrata strain SFY114 (wild-type PDR1). In both yeast species, azole upregulates Pdr1-dependent genes, including the drug efflux pumps ScPDR5 and CgCDR1i. A number of S genes that are Pdr1-dependent and azole-activated are significantly suppressed when KIX1 binds to azoles. cerevisiae as well as S. glabrata, however in S. cerevisiae, iKIX1 alone impacts distinct gene sets. cerevisiae as well as S. Glabrata. Then, either GAL11/MED15 impacts very distinct genomes in S., or iKIX1 does not dramatically change PDR1 expression. cerevisiae as well as S. Glabrata [1]. Azole effectiveness against CgPDR1 gain-of-function mutants is restored by iKIX1 (0-150 µM). In a concentration-dependent manner, it restores azole sensitivity to PDR1 gain-of-function mutants [1].
iKIX1 is an inhibitor of Pdr1-dependent gene activation. It inhibits the interaction between the KIX domain of CgGal11A and the activation domain of CgPdr1 with an IC50 of 190.2 μM and a Ki of 18 μM. It resensitizes drug-resistant C. glabrata to azole antifungals in vitro. Its activity has been confirmed in various in vitro assays.

iKIX1 resensitizes drug-resistant C. glabrata to azole antifungals in animal models for disseminated and urinary tract C. glabrata infection. It is a small molecule activator of the Pdr1 transcription factor that restores azole susceptibility in resistant Candida glabrata, demonstrated in both in vitro assays and in vivo models. It can be used to study multiple drug resistance and C. glabrata infections.

The primary in vitro assay for iKIX1 is a protein-protein interaction assay that measures its ability to inhibit the interaction between the KIX domain of CgGal11A and the activation domain of CgPdr1. This can be performed using techniques such as fluorescence polarization, surface plasmon resonance, or AlphaScreen. The IC50 of 190.2 μM and Ki of 18 μM are determined from dose-response curves in these assays.

In vitro cellular experiments for iKIX1 involve treating drug-resistant C. glabrata cells with the compound and measuring its effects on Pdr1-dependent gene expression and azole susceptibility. The expression of Pdr1 target genes, such as CDR1 and PDR5, is measured by qPCR. Azole susceptibility is assessed by determining the minimum inhibitory concentration (MIC) of fluconazole in the presence of the compound.

In vivo animal experiments for iKIX1 have been conducted in animal models of disseminated and urinary tract C. glabrata infection. The compound is administered to infected animals, and its effects on fungal burden and survival are assessed. Its ability to resensitize drug-resistant C. glabrata to azole antifungals is evaluated by combining iKIX1 with fluconazole treatment.

iKIX1 has a molecular weight of 303.17 and a molecular formula of C10H8Cl2N4OS. It has a purity of >98%. The compound is a small molecule that is typically used as a research reagent. It is an inhibitor of Pdr1-dependent gene activation and is used to study antifungal resistance mechanisms.

iKIX1 is a research compound that is not intended for human use. Its toxicological profile is not extensively characterized in the available literature. As with all research chemicals, standard safety precautions should be followed when handling. The compound is used in laboratory settings for in vitro and in vivo studies. Its safety would need to be established for therapeutic applications.

[1]. Joy L Nishikawa, et al.Inhibiting fungal multidrug resistance by disrupting an activator-Mediator interaction. Nature. 2016 Feb 25;530(7591):485-9.

iKIX1 is an antifungal agent that resensitizes drug-resistant Candida glabrata to azole antifungals in vitro and in animal models. It inhibits the interaction between the KIX domain of CgGal11A and the activation domain of CgPdr1 with an IC50 of 190.2 μM and a Ki of 18 μM. iKIX1 is used to study antifungal resistance mechanisms and C. glabrata infections.

C10H8CL2N4OS

303.17

301.979

C, 39.62; H, 2.66; Cl, 23.39; N, 18.48; O, 5.28; S, 10.58

656222-54-7

2803648

Solid powder

1.6±0.1 g/cm3

1.681

2.26

3

3

2

18

370

0

C1(C=CC(NC(NNC(CC#N)=O)=S)=CC=1Cl)Cl

XKQJVBSDCOCZFV-UHFFFAOYSA-N

InChI=1S/C10H8Cl2N4OS/c11-7-2-1-6(5-8(7)12)14-10(18)16-15-9(17)3-4-13/h1-2,5H,3H2,(H,15,17)(H2,14,16,18)

2-Cyano-acetic acid 2-[[(3,4-dichlorophenyl)amino]thioxomethyl]hydrazide

iKIX1; iKIX-1; iKIX 1;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : 61 ~83.33 mg/mL (201.2 ~274.86 mM)
Ethanol : 1 mg/mL

Solubility in Formulation 1: 2.08 mg/mL (6.86 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.86 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (6.86 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: 2.08 mg/mL (6.86 mM)

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